Compositions and methods for altering flowering and plant architecture to improve yield potential

ABSTRACT

The present invention provides recombinant DNA constructs, vectors and molecules comprising a polynucleotide sequence encoding a florigenic FT protein operably linked to a vegetative stage promoter, which may also be a meristem-preferred or meristem-specific promoter. Transgenic plants, plant cells and tissues, and plant parts are further provided comprising a polynucleotide sequence encoding a florigenic FT protein. Transgenic plants comprising a florigenic FT transgene may produce more bolls, siliques, fruits, nuts, or pods per node on the transgenic plant, particularly on the main stem of the plant, relative to a control or wild type plant. Methods are further provided for introducing a florigenic FT transgene into a plant, and planting transgenic FT plants in the field including at higher densities. Transgenic plants of the present invention may thus provide greater yield potential than wild type plants and may be planted at a higher density due to their altered plant architecture.

CROSS -REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/370,546, filed Mar. 29, 2019, which is a divisional of U.S. patentapplication Ser. No. 15/131,987, filed Apr. 18, 2016 (now U.S. Pat. No.10,294,486, issued May 21, 2019), which claims benefit of priority toU.S. Provisional Patent Application Nos. 62/150,142 and 62/233,019,filed on Apr. 20, 2015 and Sep. 25, 2015, respectively, which areincorporated herein by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of a sequence listing is filed with thisapplication by electronic submission and is incorporated into thisapplication by reference in its entirety. The sequence listing iscontained in the file named P34317US05_SL.txt, which is 61,167 bytes insize (measured in operating system MS Windows) and created on Dec. 3,2021.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for modulatingfloral development and vegetative growth by genetic modification of cropplants to increase yield.

BACKGROUND

The transition from vegetative growth to flowering is a crucial processduring plant development that is necessary for the production of grainyield in crop plants. There are four major pathways controllingflowering time in land plants that respond to environmental ordevelopmental cues, including photoperiodism (i.e., day length),vernalization (i.e., response to winter cold), and plant hormones (e.g.,gibberellins or GA), in addition to the autonomous (environmentallyindependent) pathways. Except for the GA and autonomous pathways,regulation of flowering in plants generally involves two centralregulators of flowering time, CONSTANS (CO) and FLOWERING LOCUS C (FLC).The FLC gene is a floral repressor that regulates flowering in responseto vernalization, whereas the CO gene is a floral activator thatresponds to photoperiod conditions. Under inductive photoperiodicconditions, CO activity in source leaves increases expression ofFLOWERING LOCUS T (FT), which translocates to the meristem to triggerexpression of downstream floral activating genes, including LEAFY (LFY),APETALA1 (AP1) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOCl). Othergenes, such as FLOWERING LOCUS C (FLC) and TERMINAL FLOWER 1 (TFL1), actto inhibit the expression or activity of these genes.

Except for day length neutral plants, most flowering plants respond todaily photoperiodic cycles and are classified as either short day (SD)or long day (LD) plants based on the photoperiod conditions required toinduce flowering. The photoperiod refers to the relative length orduration of light and dark periods within a 24-hour cycle. In general,long day plants tend to flower when the day length exceeds a photoperiodthreshold (e.g., as the days are getting longer in the spring), whereasshort day plants tend to flower when the day length falls below aphotoperiod threshold (e.g., as the days are getting shorter after thesummer solstice). In other words, SD plants flower as the days aregetting shorter, while LD plants flower as the days are getting longer.Soybean is an example of a short day (SD) plant in which flowering isinduced when plants are exposed to shorter daylight conditions.

Plant growers are always looking for new methods to manipulate the yieldof a plant, especially to enhance the seed yield of agronomicallyimportant crops. Thus, there is a continuing need in the art forimproved compositions and methods for increasing yields of various cropplants. It is presently proposed that improved crop yields may beachieved by enhancing agronomic traits related to flowering andreproductive development.

SUMMARY

According to a first aspect of the present invention, a recombinant DNAconstruct is provided comprising a polynucleotide sequence encoding aflorigenic FT protein operably linked to a vegetative stage promoter.The florigenic FT protein encoded by the polynucleotide sequence maycomprise an amino acid sequence having at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or least 99% identity to asequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8,10, 12, 14, 16, 18, and 20, or a functional fragment thereof. Thepolynucleotide sequence may also be at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or least 99% identity to a sequenceselected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, 17, and 19. The vegetative stage promoter may further be ameristem-preferred or meristem-specific promoter. DNA molecules andvectors comprising such a recombinant DNA construct are furtherprovided.

According to a second aspect of the present invention, transgenicplants, plant cells, plant tissues and plant parts are further providedcomprising an insertion of the recombinant DNA construct of the presentinvention into the genome of such plants, cells, tissues, and plantparts. A transgenic plant of the present invention may be homozygous orhemizygous for an insertion of the recombinant DNA construct. Atransgenic plant may be a short day plant and/or a dicotyledonous plant.Depending on the plant species, transgenic plants of the presentinvention may produce more bolls, siliques, fruits, nuts, or pods pernode of the transgenic plant, relative to a control or wild type plantnot having the recombinant DNA construct. Transgenic plants of thepresent invention may also produce more flowers and/or floral racemesper node relative to a control or wild type plant not having therecombinant DNA construct.

According to a third aspect of the present invention, methods forproducing a transgenic plant having improved yield-related traits orphenotypes are provided comprising (a) transforming at least one cell ofan explant with a recombinant DNA construct comprising a polynucleotidesequence encoding a florigenic FT protein operably linked to avegetative stage promoter; and (b) regenerating or developing thetransgenic plant from the transformed explant. Such methods may furthercomprise (c) selecting a transgenic plant having one or more of thefollowing traits or phenotypes: earlier flowering, longer reproductiveor flowering duration, increased number of flowers per node, increasednumber of floral racemes per node, increased number of pods, bolls,siliques, fruits, or nuts per node, and increased number of seeds pernode, as compared to a control plant not having the recombinant DNAconstruct.

According a fourth aspect of the present invention, methods are providedfor planting a transgenic crop plant of the present invention at anormal or higher density in the field. According to some embodiments,methods are provided comprising: planting a transgenic crop plant at ahigher density in the field, wherein the transgenic crop plant istransformed with a recombinant DNA construct comprising a polynucleotidesequence encoding a florigenic FT protein operably linked to avegetative stage promoter. According to some of these embodiments, thevegetative stage promoter may be a meristem-preferred ormeristem-specific promoter. For soybean, a higher density of about150,000 to 250,000 seeds of the transgenic soybean plant may be plantedper acre. For cotton, a higher density of about 48,000 to 60,000 seedsof the transgenic cotton plant may be planted per acre. For canola, ahigher density of about 450,000 to 680,000 seeds of the transgeniccanola plant may be planted per acre.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a matrix table showing a comparison of nucleotidesequences for each combination of the various FT genes including theirpercent identity.

FIG. 1B provides a matrix table showing a comparison of proteinsequences for each combination of the various FT proteins includingtheir percent identity.

FIG. 1C provides a CLUSTAL 2.0.9 multiple sequence alignment of variousFT proteins identified as Gm.FT2a with SEQ ID NO: 2, Gm.FT2b with SEQ IDNO: 4, Le.FT with SEQ ID NO: 12, Pt.FT with SEQ ID NO: 20, Os.HD3a withSEQ ID NO: 18, At.FT with SEQ ID NO: 14, At. TSF with SEQ ID NO: 16,Nt.FT with SEQ ID NO: 10, Gm.FT5a with SEQ ID NO: 6 and Zm.ZCN8 with SEQID NO: 8.

FIG. 2 shows the total FT transcript levels in soybean leaf and apextissues collected at 1, 3 and 5 days after either a short day or longday light treatment.

FIGS. 3A to 3O and FIGS. 4A to 4O show the expression pattern of thepAt.Erecta promoter by monitoring GUS activity during early soybeandevelopment. FIGS. 3A to 3O are a set of black and white images ofstained tissues, and the images in FIGS. 4A to 4O correspond to FIGS. 3Ato 3O but are filtered for blue GUS staining. FIGS. 3A to 3C and 4A to4C show expression in a 3-day-old germinating seedling; FIGS. 3D to 3Iand 4D to 4I show expression in a 10-day-old vegetative shoot (grown in14 hour light/10 hour dark photoperiod); FIGS. 3J to 3L and 4J to 4Lshow expression in a 16-day-old reproductive shoot; and FIGS. 3M to 3Oand 4M to 4O show expression in the 30d old mature and immature leavesof the reproductive shoot. Bars are 100₁1.m.

FIGS. 5A to 5F and FIGS. 6A to 6F show the GUS expression pattern withthe pAT.Erecta promoter during R1 and floral stages of development(35-40 days after germination). FIGS. 5A to 5F are a set of black andwhite images of stained tissues, and the images in FIGS. 6A to 6Fcorrespond to FIGS. 5A to 5F but are filtered for blue GUS staining.FIGS. 5A and 6A show expression in the inflorescence stems or pedicels(arrows), and FIGS. 5B and 6B show expression in the floral peduncle(arrows). Expression is also shown in the vasculature and parenchymacells (FIGS. 5C and 6C), in stamen filaments (FIGS. 5D and 6D; arrow),and unpollinated ovules (FIGS. 5E, 5F, 6E and 6F; arrows). Bars are 1mm.

FIG. 7 shows section imaging of the shoot apical meristem (SAM) fromwild type versus GmFT2a-expressing transgenic plants at 7 days afterplanting using scanning electron microscopy (eSEM) analysis.

FIG. 8 shows scanning electron microscopy (eSEM) micrographs of anaxillary inflorescence primordia from a wild type plant (collected at 27days after planting), in comparison to an axillary inflorescenceprimordia from a transgenic event expressing Gm.FT2a (collected at 9days after planting).

FIGS. 9A to 9C show the effects of Gm.FT2a expression driven by theAt.Erecta promoter in soybean. FIG. 9A depicts a null segregant showingnormal axillary buds, whereas FIG. 9B and FIG. 9C (corresponding toplants homozygous or hemizygous for the Gm.FT2a transgene, respectively)each show early flowering and increased pods per node relative to thenull segregant.

FIG. 10 shows a whole plant image of a wild type null segregant next toplants hemizygous and homozygous for the Gm.FT2a transgene as indicated.

FIG. 11 shows images of the main stem of plants that are homozygous orhemizygous for the pAt.Erecta-Gm.FT2a transgene in comparison to a nullsegregant as indicated.

FIG. 12A shows whole plant images of a wild type null segregant and aplant homozygous for the pEr:Zm.ZCN8 transgene as indicated.

FIG. 12B shows close up images of pods on the mainstem of a wild typenull segregant and a plant homozygous for the pEr:Zm.ZCN8 transgene asindicated.

FIG. 13A shows whole plant images of a wild type null segregant and aplant homozygous for the pEr:Nt.FT-like transgene as indicated.

FIG. 13B shows close up images of pods on the mainstem of a wild typenull segregant and a plant homozygous for the pEr:Nt.FT-like transgeneas indicated.

FIG. 14 shows whole plant images of a wild type null segregant and aplant homozygous for the pEr:Gm.FT2b transgene as indicated.

FIG. 15 shows whole plant images of a wild type null segregant and aplant homozygous for the pEr:Le.SFT transgene as indicated.

FIG. 16 shows whole plant images of a wild type null segregant and aplant homozygous for the pEr:FT5a transgene as indicated.

DETAILED DESCRIPTION

The goal of improving yield is common to all crops across agriculture.The present invention includes methods and compositions for improvingyield in flowering (angiosperm) or seed-bearing plants by modificationof traits associated with flowering time, reproductive development, andvegetative growth to improve one or more flowering and/or yield-relatedtraits or phenotypes, such as the number of flowers, seeds and/or podsper plant, and/or the number of flowers, seeds and/or pods per node(and/or per main stem) of the plant. Without being bound by any theory,compositions and methods of the present invention may operate to improveyield of a plant by increasing the number of floral meristems,increasing synchronization of lateral meristem release, and/or extendingthe time period for pod or seed development in the plant.

Previously, it was discovered that growing short day plants, such assoybean, under long day conditions (e.g., about 14-16 hours of light perday) and then briefly subjecting those plants to short day growingconditions (e.g., about 9-11 hours of light per day for about 3-21 days)before returning the plants to long day (non-inductive) growingconditions, produced plants having increased numbers of pods/seeds perplant (and pods/seeds per node and/or per branch). See, e.g., U.S. Pat.No. 8,935,880 and U.S. Patent Application Publication No. 2014/0259905,the entire contents and disclosures of which are incorporated herein byreference.

As described further below, this short day induction phenotype insoybean was used to identify genes having altered expression in theseplants through transcriptional profiling. These studies identifiedseveral genes with altered expression in these treated soybean plantsincluding an endogenous FT gene, Gm.FT2a, having increased expression inresponse to the short day induction treatment. Thus, it is presentlyproposed that transgenic FT expression as described herein may be usedin place of short day induction to increase seed yield, alterreproductive traits or phenotypes in plants, or both. According to anaspect of the present invention, ectopic or transgenic expression of aGm.FT2a gene or other FT sequence, or a functional fragment, homolog orortholog thereof, in a flowering or seed-bearing plant may be used toincrease seed yield and/or alter one or more reproductive phenotypes ortraits, which may involve an increase in the number of pods/seeds perplant (and/or the number of pods/seeds per node or main stem of theplant). As explained further below and depending on the particular plantspecies, these yield-related or reproductive phenotypes or traits mayalso apply to other botanical structures analogous to pods of leguminousplants, such as bolls, siliques, fruits, nuts, tubers, etc. Thus, aplant ectopically expressing a FT sequence may instead have an increasednumber of bolls, siliques, fruits, nuts, tubers, etc., per node(s), mainstem, and/or branch(es) of the plant, and/or an increased number ofbolls, siliques, fruits, nuts, tubers, etc., per plant.

According to embodiments of the present invention, a recombinant DNAmolecule comprising an FT transgene is provided, which may be used intransformation to generate a transgenic plant expressing the FTtransgene. The polynucleotide coding sequence of the FT transgene mayinclude Gm.FT2a (SEQ ID NO: 1), or any polynucleotide sequence encodingthe Gm.FT2a protein (SEQ ID NO: 2). The polynucleotide coding sequenceof an FT transgene may also correspond to other FT genes in soybean orother plants. For example, other polynucleotide FT coding sequences fromsoybean that may be used according to present embodiments include:Gm.FT5a (SEQ ID NO: 3) or a polynucleotide encoding a Gm.FT5a protein(SEQ ID NO: 4), or Gm.FT2b (SEQ ID NO: 5) or a polynucleotide encoding aGm.FT2b protein (SEQ ID NO: 6). In addition, examples of polynucleotideFT coding sequences from other plant species that may be used include:Zm.ZCN8 (SEQ ID NO: 7) from maize or a polynucleotide encoding Zm.ZCN8protein (SEQ ID NO: 8), Nt.FT-like or Nt.FT4 (SEQ ID NO: 9) from tobaccoor a polynucleotide encoding Nt.FT-like or Nt.FT4 protein (SEQ ID NO:10), Le.FT or SFT (SEQ ID NO: 11) from tomato or a polynucleotideencoding Le.FT or SFT protein (SEQ ID NO: 12), At.FT (SEQ ID NO: 13)from Arabidopsis or a polynucleotide encoding At.FT protein (SEQ ID NO:14), At. TSF (SEQ ID NO: 15) from Arabidopsis or a polynucleotideencoding At.TSF protein (SEQ ID NO: 16), Os.HD3a (SEQ ID NO: 17) fromrice or a polynucleotide encoding Os.HD3a protein (SEQ ID NO: 18), orPt.FT (SEQ ID NO: 19) from Populus trichocarpa or a polynucleotideencoding Pt.FT protein (SEQ ID NO: 20).

Polynucleotide coding sequences for FT transgenes encoding additional FTproteins from other species having known amino acid sequences may alsobe used according to embodiments of the present invention, which may,for example, include the following: Md.FT1 and Md.FT2 from apple (Malusdomestica); Hv.FT2 and Hv.FT3 from barley (Hordeum vulgare); Cs.FTL3from Chrysanthemum; Ls.FT from lettuce (Lactuca sativa); Pn.FT1 andPn.FT2 from Lombardy poplar (Populus nigra); Pa.FT from London planetree (Platanus acerifolia); Dl.FT1 from Longan (Dimocarpus longan);Ps.FTa1, Ps.FTa2, Ps.FTb 1, Ps.FTb2, and Ps.FTc from pea (Pisumsativum); Ac.FT from pineapple (Ananas comosus); Cm-FTL1 and Cm-FTL2from pumpkin (Cucurbita maxima); Ro.FT from rose; Cg.FT from springorchid (Cymbidium); Fv.FT1 from strawberry (Fragaria vesca); Bv.FT2 fromsugar beet (Beta Vulgaris); Ha.FT4 from sunflower (Helianthus annuus);and Ta.FT or TaFT1 from wheat (Triticum aestivum). See, e.g., Wickland,DP et al., “The Flowering Locus T/Terminal Flower 1 Gene Family:Functional Evolution and Molecular Mechanisms”, Molecular Plant 8:983-997 (2015), the content and disclosure of which is incorporatedherein by reference.

Unless otherwise stated, nucleic acid or polynucleotide sequencesdescribed herein are provided (left-to-right) in the 5′ to 3′ direction,and amino acid or protein sequences are provided (left-to-right) in theN-terminus to C-terminus direction. Additional known or later discoveredFT genes and proteins from these or other species may also be usedaccording to embodiments of the present invention. These FT genes may beknown or inferred from their nucleotide and/or protein sequences, whichmay be determined by visual inspection or by use of a computer-basedsearching and identification tool or software (and database) based on acomparison algorithm with known FT sequences, structural domains, etc.,and according to any known sequence alignment technique, such as BLAST,FASTA, etc.

According to embodiments of the present invention, an FT transgene of arecombinant DNA molecule may comprise a polynucleotide sequence that(when optimally aligned) is at least 60% identical, at least 65%identical, at least 70% identical, at least 75% identical, at least 80%identical, at least 85% identical, or at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or least 99% identical to one or more of thepolynucleotide FT coding sequences listed above (e.g., SEQ ID NOS: 1, 3,5, 7, 9, 11, 13, 15, 17, or 19), or to any other known florigenic FTcoding sequence. Sequence identity percentages among polynucleotidesequences of the above listed full length FT genes are presented in FIG.1A. Each cell in the table in FIG. 1A shows the percentage identity forthe FT gene in the corresponding row (query sequence) as compared to theFT gene in the corresponding column (subject sequence) divided by thetotal length of the query sequence, and the number in parenthesis is thetotal number of identical bases between the query and subject sequences.As shown in this figure, the percentage identities among polynucleotidesequences for these sampled FT genes range from about 60% to about 90%identity. Thus, a polynucleotide sequence that is within one or more ofthese sequence identity ranges or has a higher sequence identity may beused according to embodiments of the present invention to induceflowering, increase yield, and/or alter one or more reproductive traitsof a plant. Similar polynucleotide coding sequences for FT may bedesigned or chosen based on known FT protein sequences, conserved aminoacid residues and domains, the degeneracy of the genetic code, and anyknown codon optimizations for the particular plant species to betransformed.

As described below, an FT transgene comprising any one of the abovecoding sequences may further include one or more expression and/orregulatory element(s), such as leader(s), intron(s), etc. Indeed, an FTtransgene may comprise a genomic sequence encoding an FT protein oramino acid sequence, or a fragment or portion thereof.

According to embodiments of the present invention, an FT transgene of arecombinant DNA molecule may comprise a polynucleotide sequence encodingan amino acid or protein sequence that (when optimally aligned) is atleast 60% identical, at least 65% identical, at least 70% identical, atleast 75% identical, at least 80% identical, at least 85% identical, orat least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or least 99%identical to any one or more of the FT protein or amino acid sequenceslisted above (e.g., SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20)or any other known florigenic FT protein sequence, or a functionalfragment thereof. Such a “functional fragment” is defined as a proteinhaving a polypeptide sequence that is identical or highly similar to afull-length FT protein but lacking one or more amino acid residues,portions, protein domains, etc., of the full-length FT protein, as longas the fragment remains active in causing one or more of the phenotypiceffects or changes similar to the full-length protein whentransgenically expressed in a plant. Sequence identity percentages amongthe above listed full length FT proteins are presented in FIG. 1B. Thepercentages are calculated as described above in reference to FIG. 1Abased on the number of identical amino acid residues (in parenthesis)between the query and subject FT protein sequences. Multiple sequencealignment of these FT proteins is also shown in FIG. 1C. As can be seenfrom these figures, the percentage identity among protein sequences forthese FT genes ranges from about 60% to about 90% identity. Thus, apolynucleotide sequence encoding an amino acid or protein sequence thatis within one or more of these sequence identity ranges or has a highersequence identity may be used according to embodiments of the presentinvention to induce flowering, increase seed yield, and/or alter one ormore reproductive traits of a plant. These FT protein sequences encodedby a polynucleotide sequence of the present invention may be designed orchosen based on known FT protein sequences and their conserved aminoacid residues and domains.

As used herein, the term “sequence identity” refers to the extent towhich two optimally aligned DNA sequences are identical. Variouspair-wise or multiple sequence alignment algorithms and programs areknown in the art, such as ClustalW, etc., that may be used to comparethe sequence identity or similarity between two or more sequences, suchas between two or more FT genes or protein sequences, or an FT gene(nucleotide) or protein sequence and another nucleotide or proteinsequence. For example, the percentage identity of one sequence (query)to another sequence (subject) may be calculated as described above inreference to FIGS. 1A and 1B (i.e., with the sequences optimallyaligned, divide the number of identical bases or residues by the totalnumber of bases or residues for the query sequence, and multiply by100%). Although other alignment and comparison methods are known in theart, the alignment and percent identity between two sequences (includingthe percent identity ranges described above) may be as determined by theClustalW algorithm, see, e.g., Chenna R. et al., “Multiple sequencealignment with the Clustal series of programs,” Nucleic Acids Research31: 3497-3500 (2003); Thompson J D et al., “Clustal W: Improving thesensitivity of progressive multiple sequence alignment through sequenceweighting, position-specific gap penalties and weight matrix choice,”Nucleic Acids Research 22: 4673-4680 (1994); and Larkin M A et al.,“Clustal W and Clustal X version 2.0,” Bioinformatics 23: 2947-48(2007), the entire contents and disclosures of which are incorporatedherein by reference. For purposes of the present invention, when twosequences are optimally aligned (with allowance for gaps in theiralignment), then the “percent identity” for the query sequence iscalculated as described above in reference to FIGS. 1A and 1B—i.e.,Percent Identity=(Number of Identical Positions between query andsubject sequences/Total Number of Positions in the Query Sequence)×100%,with each sequence consisting of a series of positions (nucleotide basesor amino acid residues).

An FT protein sequence encoded by a polynucleotide sequence or transgeneof the present invention may also be designed or chosen to have one ormore amino acid substitution(s) known to be chemically and/orstructurally conservative (e.g., replacing one amino acid with anotherhaving similar chemical or physical properties, such as hydrophobicity,polarity, charge, steric effect, acid/base chemistry, similar side chaingroup, such as hydroxyl, sulfhydryl, amino, etc.) to avoid or minimizestructural changes to the protein that might affect its function. Forexample, valine is often a conservative substitute for alanine, andthreonine may be a conservative substitute for serine. Additionalexamples of conservative amino acid substitutions in proteins include:valine/leucine, valine/isoleucine, phenylalanine/tyrosine,lysine/arginine, aspartic acid/glutamic acid, and asparagine/glutamine.An FT protein sequence encoded by a polynucleotide sequence or transgeneof the present invention may also include proteins that differ in one ormore amino acids from those of a known FT protein or similar sequence asa result of deletion(s) and/or insertion(s) involving one or more aminoacids.

Various FT genes and proteins from different plant species may beidentified and considered FT homologs or orthologs for use in thepresent invention if they have a similar nucleic acid and/or proteinsequence and share conserved amino acids and/or structural domain(s)with at least one known FT gene or protein. As used herein, the term“homolog” in reference to a FT gene or protein is intended tocollectively include any homologs, analogs, orthologs, paralogs, etc.,of the FT gene or protein, and the term “homologous” in reference topolynucleotide or protein sequences is intended to mean similar oridentical sequences. Such a FT homolog may also be defined as having thesame or similar biological function as known FT genes (e.g., acting tosimilarly influence flowering and/or other reproductive or yield-relatedtraits or phenotypes when ectopically expressed in a plant).

Sequence analysis and alignment of FT protein sequences from differentplant species further reveals a number of conserved amino acid residuesand at least one conserved structural domain. By subjecting the variousaligned FT protein sequences (see, e.g., FIGS. 1B and 1C) to a proteindomain identification tool using a Pfam database (e.g., Pfam version26.0, released November 2011, or later version), these FT proteins havebeen found to contain and share at least a portion of a putativephosphatidyl ethanolamine-binding protein (PEBP) domain (Pfam domainname: PBP_N; Accession number: PF01161). See, e.g., Banfield, M J etal., “The structure of Antirrhinum centroradialis protein (CEN) suggestsa role as a kinase inhibitor,” Journal of Mol Biol., 297(5): 1159-1170(2000), the entire contents and disclosure of which are incorporatedherein by reference. This PEBP domain was found to correspond, forexample, to amino acids 28 through 162 of the full length Gm.FT2aprotein (See Table 5 below). Thus, FT proteins encompassed byembodiments of the present invention may include those identified orcharacterized as having or containing at least a PEBP domain (Accessionnumber: PF01161) according to Pfam analysis. Accordingly, the presentinvention may further include a polynucleotide sequence(s) encoding anFT protein having at least a PEBP domain. As known in the art, the“Pfam” database is a large collection of multiple sequence alignmentsand hidden Markov models covering many common protein families andcontaining information about various protein families and their domainstructure(s). By identifying a putative Pfam structural domain(s) for agiven protein sequence, the classification and function of the proteinmay be inferred or determined. See, e.g., Finn, R D et al., “The Pfamprotein families database,” Nucleic Acids Research (Database Issue),42:D222-D230 (2014), the entire contents and disclosure of which areincorporated herein by reference.

Embodiments of the present invention may further include polynucleotidesequence(s) encoding inductive or florigenic FT proteins. An FT proteinencoded by a polynucleotide sequence may be “inductive” or “florigenic”if the FT protein, when ectopically expressed in a plant, is able tocause earlier flowering and/or an increased prolificacy in the number offlowers, pods, and/or seeds per one or more node(s) of the plant.Without being bound by any theory, such increased prolificacy in thenumber of flowers, pods, and/or seeds per node(s) of the plant mayresult from an increase in the number of meristems at those node(s) thatundergo a vegetative to reproductive transition and produce flowers.Such an increased prolificacy at each node due to ectopic expression ofa “florigenic” FT may be due to increased synchronization of the releaseand floral development of early racemes and lateral meristems at eachnode. Although a “florigenic” FT protein may function to induce earlierflowering when ectopically expressed in a plant, a transgenicallyexpressed “florigenic” FT protein may increase the number of flowers,pods, and/or seeds per node(s) of a plant through one or more pathwaysor mechanisms that are independent of, or in addition to, any florigeniceffects related to flowering time and/or reproductive duration.

Florigenic FT-like genes from various plant species are generally wellconserved. However, many proteins in the PEBP family have amino acidsequences that are substantially similar to florigenic FT proteins butdo not behave as florigens. For example, Terminal Flower (TFL) genesfrom various plant species have similar protein sequences to florigenicFT genes but actually delay flowering. Recent work has identifiedspecific amino acid residues that are generally not shared betweenflorigenic FT proteins and other PEBP proteins, such as TFLs, andsubstitutions at many of these positions have been shown to convertflorigenic FT proteins into floral repressor proteins. See, e.g., Ho andWeigel, Plant Cell 26: 552-564 (2014); Danilevskaya et al., PlantPhysiology 146(1): 250-264 (2008); Harig et al., Plant Journal 72:908-921 (2012); Hsu et al., Plant Cell 18: 1846-1861 (2006); Kojima etal., Plant Cell Physiology 43(10): 1096-1105 (2002); Kong et al., PlantPhysiology 154: 1220-1231 (2010); Molinero-Rosales et al., Planta 218:427-434 (2004); Zhai et al., PLoS ONE, 9(2): e89030 (2014), and WicklandD P et al. (2015), supra, the entire contents and disclosures of whichare incorporated herein by reference. Thus, these amino acid residuescan serve as signatures to further define and distinguish florigenic FTproteins of the present invention.

According to embodiments of the present invention, an “inductive” or“florigenic” FT protein may be further defined or characterized ascomprising one or more of the following amino acid residue(s) (aminoacid positions refer to corresponding positions of the full-lengthArabidopsis FT protein, SEQ ID NO: 14): a proline at amino acid position21 (P21); an arginine or lysine at amino acid position 44 (R44 or K44);a glycine at amino acid position 57 (G57); a glutamic acid or anaspartic acid at amino acid position 59 (E59 or D59); a tyrosine atamino acid position 85 (Y85); a leucine at amino acid position 128(L128); a glycine at amino acid position 129 (G129); a threonine atamino acid position 132 (T132); an alanine at amino acid position 135(A135); a tryptophan at amino acid position 138 (W138); a glutamic acidor an aspartic acid at amino acid position 146 (E146 or D146); and/or acysteine at amino acid position 164 (C164). Corresponding amino acidpositions of other FT proteins can be determined by alignment with theArabidopsis FT sequence (see, e.g., FIG. 1C). One skilled in the artwould be able to identify corresponding amino acid positions of other FTproteins based on their sequence alignment. Several of these keyresidues fall within an external loop domain of FT-like proteins,defined as amino acids 128 through 145 of the Arabidopsis full-length FTsequence (SEQ ID NO: 14) and corresponding sequences of other FTproteins (see, e.g., FIG. 1C). Thus, polynucleotides of the presentinvention may encode florigenic FT proteins having one or more of theseconserved amino acid residues.

Florigenic FT proteins of the present invention may also have one ormore other amino acids at one or more of the above identified residuepositions. For example, in reference to the above amino acid positionsof the Arabidopsis FT (At.FT) protein sequence (SEQ ID NO: 14), aflorigenic FT protein may alternatively have one or more of thefollowing amino acids: an alanine (in place of proline) at the positioncorresponding to position 21 of the At.FT protein sequence (P21A), orpossibly other small, nonpolar residues, such as glycine or valine, atthis position; a histidine (in place of lysine or arginine) at the aminoacid position corresponding to position 44 of the At.FT proteinsequence, or possibly other polar amino acids at this position; analanine or cysteine (in place of glycine) at the amino acid positioncorresponding to position 57 of the At.FT protein sequence, or possiblyother small, nonpolar residues, such proline or valine, at thisposition; an asparagine or serine (in place of glutamic acid or asparticacid) at the amino acid position corresponding to position 59 of theAt.FT protein sequence, or possibly other small, polar residues, such asglutamine, cysteine, or threonine, at this position; a variety of polarand nonpolar uncharged residues (other than tyrosine) at the amino acidposition corresponding to position 85 of the At.FT protein sequence; anonpolar or hydrophobic uncharged residue (other than leucine), such asisoleucine, valine, or methionine, at the amino acid positioncorresponding to position 128 of the At.FT protein sequence; a varietyof smaller nonpolar and uncharged residues (other than glycine), such asalanine, valine, leucine, isoleucine, methionine, etc., at the aminoacid position corresponding to position 129 of the At.FT proteinsequence, although some polar and charged residues may be tolerated atthis position; a polar uncharged residue (other than threonine) at theamino acid position corresponding to position 132 of the At.FT proteinsequence; a variety of amino acids other than proline , such asthreonine, at the amino acid position corresponding to position 135 ofthe At.FT protein sequence ; a variety of other bulky nonpolar orhydrophobic amino acids (in place of tryptophan), such as methionine orphenylalanine, at the amino acid position corresponding to position 138of the At.FT protein sequence; a variety of other polar ornon-positively charged amino acids , such as asparagine or serine, atthe amino acid position corresponding to position 146 of the At.FTprotein sequence; and/or a variety of other polar or nonpolar aminoacids (in place of cysteine, such as isoleucine, at the amino acidposition corresponding to position 164 of the At.FT protein sequence.One skilled in the art would be able to identify corresponding aminoacid positions and substitutions of FT proteins based on their sequencealignment to the Arabidopsis FT protein sequence. In addition, otherchemically conservative amino acid substitutions are also contemplatedwithin the scope of florigenic FT proteins based on the knowledge of oneskilled in the art of protein biochemistry. Accordingly, polynucleotidesof the present invention may further encode florigenic FT proteinshaving one or more conservative amino acid substitutions. Indeed,florigenic FT proteins encoded by polynucleotides of the presentinvention include native sequences and artificial sequences containingone or more conservative amino acid substitutions, as well as functionalfragments thereof.

Florigenic FT proteins of the present invention may also be defined asexcluding (i.e., not having) one or more amino acid substitutions thatmay be characteristic of, or associated with, TFL or othernon-florigenic or anti-florigenic proteins. For example, in reference tothe amino acid positions of the Arabidopsis FT protein sequence (SEQ IDNO: 14), a florigenic FT protein may exclude one or more of thefollowing amino acids: a phenylalanine or serine at the positioncorresponding to position 21 of the At.FT protein sequence (e.g., inplace of proline or alanine); a phenylalanine at the positioncorresponding to position 44 of the At.FT protein sequence (e.g., inplace of arginine or lysine); a histidine, glutamic acid, or asparticacid at the position corresponding to position 57 of the At.FT proteinsequence (e.g., in place of glycine); a glycine or alanine at theposition corresponding to position 59 of the At.FT protein sequence(e.g., in place of glutamic acid or aspartic acid); a histidine at theposition corresponding to position 85 of the At.FT protein sequence(e.g., in place of tyrosine); a lysine, arginine, alanine, or methionineat the position corresponding to position 109 of the At.FT proteinsequence; a lysine or arginine at the position corresponding to position128 of the At.FT protein sequence (e.g., in place of leucine); aglutamine or asparagine at the position corresponding to position 129 ofthe At.FT protein sequence (e.g., in place of glycine); a valine orcysteine at the position corresponding to position 132 of the At.FTprotein sequence (e.g., in place of threonine); a lysine, arginine, oralanine at the position corresponding to position 134 of the At.FTprotein sequence (e.g., in place of tyrosine); a proline at the positioncorresponding to position 135 of the At.FT protein sequence (e.g., inplace of alanine or threonine); a serine, aspartic acid, glutamic acid,alanine, lysine, or arginine at the position corresponding to position138 of the At.FT protein sequence (e.g., in place of tryptophan ormethionine); a lysine or arginine at the position corresponding toposition 140 of the At.FT protein sequence; a lysine or arginine at theposition corresponding to position 146 of the At.FT protein sequence(e.g., in place of acidic or uncharged polar residues); a lysine orarginine at the position corresponding to position 152 of the At.FTprotein sequence; and/or an alanine at the position corresponding toposition 164 of the At.FT protein sequence (e.g., in place of cysteineor isoleucine). One skilled in the art would be able to identifycorresponding amino acid positions and substitutions of other FTproteins based on their sequence alignment. Accordingly, embodiments ofthe present invention may exclude polynucleotides that encode FT-likeproteins having one or more of the above amino acid substitutionsassociated with TFL or other anti-florigens. However, an FT protein maytolerate one or some of these amino acid substitutions while stillmaintaining florigenic activity.

A florigenic FT protein of the present invention may also be defined asbeing similar to a known FT protein in addition to having one or more ofthe above signature amino acid residues. For example, a florigenicprotein may be defined as having at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or least 99% identity to a sequenceselected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, and 20, or a functional fragment thereof, in addition to oneor more of the following signature residues: a tyrosine or otheruncharged polar or nonpolar residue (e.g., alanine, tryptophan,methionine, leucine, threonine, cysteine, serine, or asparagine) at theamino acid position corresponding to position 85 of the At.FT proteinsequence; a leucine or other nonpolar or hydrophobic residue (e.g.,isoleucine, valine, or methionine) at the amino acid positioncorresponding to position 128 of the At.FT protein sequence; and/or atryptophan or other large nonpolar or hydrophobic residue (e.g.,methionine or phenylalanine) at the amino acid position corresponding toposition 138 of the At.FT protein sequence. Such a florigenic FT proteinmay be further defined as having additional signature amino acidresidue(s), such as one or more of the following: a glycine or othersmall nonpolar and uncharged residue (e.g., alanine, valine, leucine,isoleucine, or methionine) at the amino acid position corresponding toposition 129 of the At.FT protein sequence; and/or a threonine at theamino acid position corresponding to position 132 of the At.FT proteinsequence.

A florigenic FT protein of the present invention may also be defined ashaving at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or least 99% identity to a sequence selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, or afunctional fragment thereof, but not having (i.e., excluding) one ormore non-florigenic or anti-florigenic residues, such as one or more ofthe following: a histidine at the amino acid position corresponding toposition 85 of the At.FT protein sequence; a lysine or arginine at theamino acid position corresponding to position 128 of the At.FT proteinsequence; and/or a serine, aspartic acid, glutamic acid, lysine orarginine at the amino acid position corresponding to position 138 of theAt.FT protein sequence. Such a florigenic FT protein may be furtherdefined as not having (i.e., excluding) one or more additional residues,such as one or more of the following: a glutamine or asparagine at theamino acid position corresponding to position 129 of the At.FT proteinsequence; and/or a valine or cysteine at the amino acid positioncorresponding to position 132 of the At.FT protein sequence.

Flowering Locus T (FT) genes play a key role in higher plants andfunction to integrate floral pathways. FT proteins have been shown tofunction as a mobile signal or florigen transported from leaves to theshoot apical apex where it triggers initiation of reproductivedevelopment in diverse species. See, e.g., Jaeger, K. E. et al.,“Interlocking feedback loops govern the dynamic behavior of the floraltransition in Arabidopsis,” The Plant Cell, 25:820-833 (2013);Corbesier, L et al., “FT protein movement contributes to long distancesignaling in floral induction of Arabidopsis,” Science 316: 1030-1033(2007); Jaeger, K E et al., “FT protein acts as a long range signal inArabidopsis,” Curr Biol 17: 1050-1054 (2007); and Amasino, R. M. et al.,“The Timing of Flowering,” Plant Physiology, 154(2):516-520 (2010), theentire contents and disclosures of which are incorporated herein byreference. In Arabidopsis, FT protein binds to 14-3-3 and FloweringLocus D (FD) proteins in the meristem to form a flowering complextriggering activation of key floral meristem identity genes, such asAPETATALI (API) and SOCl at the shoot apex. See, e.g., Taoka, K. et al.,“14-3-3 protein act as intracellular receptors for rice Hd3a florigen.”Nature 476:332-335 (2011). The TERMINAL FLOWER 1 (TFL1) gene is a keyrepressor of FT targets that maintains the center of the shoot apicalmeristem (SAM) in a vegetative state. TFL1 acts by repressing the LEAFY(LFY) and API genes. Thus, the relative concentrations of FT and TFL1 inthe target tissues act competitively to control the timing of thereproductive transition of meristems from a vegetative state that mayterminate further vegetative growth. . See, e.g., Abe, M et al., Science309:1052-1055 (2005); and McGarry, RC et al., Plant Science 188/189:71-81 (2012).

FT genes have been identified from many diverse species, and ectopic FTexpression has been reported to induce early flowering. See, e.g., Kong,F. et al., “Two Coordinately Regulated Homologs of Flowering Locus T AreInvolved in the Control of Photoperiodic Flowering in Soybean,” PlantPhysiology 154: 1220-1231 (2010); Turck, F. et al., “Regulation andidentity of florigen: Flowering Locus T moves center stage,” Ann RevPlant Biol 59: 573-594 (2008); Blackman, B K et al., “The role ofrecently derived FT paralogs in sunflower domestication,” Curr Biol 20:629-635 (2010); Lifschitz, E. et al., “The tomato FT orthologs triggerssystemic signals that regulate growth and flowering and substitute fordiverse environmental stimuli,” PNAS 103: 6398-6403 (2006); Trankner, C.et al., “Over-expression of an FT-homologous gene of apple induces earlyflowering in annual and perennial plants,” Planta 232: 1309-1324 (2010);and Xiang, L. et al., “Functional analysis of Flowering Locus Torthologs from spring orchid (Cymbidium goeringii Rchb. f.) thatregulates the vegetative to reproductive transition,” Plant Cell &Biochem 58: 98-105 (2012), the entire contents and disclosures of whichare incorporated herein by reference. However, prior studies withexpression of FT transgenes used constitutive or tissue specificpromoters that produced either very severe phenotypes, non-cellautonomous (systemic) phenotypes, or autonomous leaf specific phenotypeswith plants or seedlings flowering earlier than controls and terminatingat early stages of development. Given these findings, ectopic FTexpression was generally not seen as a viable approach to increasingyield in plants by inducing flowers or altering flowering time.

According to embodiments of the present invention, a recombinant DNAmolecule or polynucleotide is provided comprising a polynucleotidecoding sequence encoding a FT protein that is operably linked to one ormore promoter(s) and/or other regulatory element(s) that are operable ina plant cell to control or bias the timing and/or location of FTexpression when transformed into a plant. As commonly understood in theart, the term “promoter” may generally refer to a DNA sequence thatcontains an RNA polymerase binding site, transcription start site,and/or TATA box and assists or promotes the transcription and expressionof an associated transcribable polynucleotide sequence and/or gene (ortransgene). A promoter may be synthetically produced, varied or derivedfrom a known or naturally occurring promoter sequence or other promotersequence (e.g., as provided herein). A promoter may also include achimeric promoter comprising a combination of two or more heterologoussequences. A promoter of the present invention may thus include variantsof promoter sequences that are similar in composition, but not identicalto, other promoter sequence(s) known or provided herein. As used herein,the term “operably linked” refers to a functional linkage between apromoter or other regulatory element and an associated transcribablepolynucleotide sequence or coding sequence of a gene (or transgene),such that the promoter, etc., operates to initiate, assist, affect,cause, and/or promote the transcription and expression of the associatedcoding or transcribable sequence, at least in particular tissue(s),developmental stage(s), and/or under certain condition(s).

A promoter may be classified according to a variety of criteria relatingto the pattern of expression of a coding sequence or gene (including atransgene) operably linked to the promoter, such as constitutive,developmental, tissue-specific, inducible, etc. Promoters that initiatetranscription in all or most tissues of the plant are referred to as“constitutive” promoters. Promoters that initiate transcription duringcertain periods or stages of development are referred to as“developmental” promoters. Promoters whose expression is enhanced incertain tissues of the plant relative to other plant tissues arereferred to as “tissue-enhanced” or “tissue-preferred” promoters. Thus,a “tissue-preferred” promoter causes relatively higher or preferentialexpression in a specific tissue(s) of the plant, but with lower levelsof expression in other tissue(s) of the plant. Promoters that expresswithin a specific tissue(s) of the plant, with little or no expressionin other plant tissues, are referred to as “tissue-specific” promoters.A promoter that expresses in a certain cell type of the plant isreferred to as a “cell type specific” promoter. An “inducible” promoteris a promoter that initiates transcription in response to anenvironmental stimulus such as cold, drought or light, or other stimuli,such as wounding or chemical application. A promoter may also beclassified in terms of its origin, such as being heterologous,homologous, chimeric, synthetic, etc. A “heterologous” promoter is apromoter sequence having a different origin relative to its associatedtranscribable sequence, coding sequence, or gene (or transgene), and/ornot naturally occurring in the plant species to be transformed. The term“heterologous” may refer more broadly to a combination of two or moreDNA molecules or sequences when such a combination is not normally foundin nature.

According to embodiments of the present invention, a recombinant DNAmolecule or polynucleotide is provided comprising a florigenic FTtransgene or coding sequence operably linked to a promoter thatfunctions in a plant, which may be introduced or transformed into aplant to cause the plant to have an altered flowering, reproductiveand/or yield-related trait or phenotype. Since FT proteins are believedto operate in the meristems of a plant, including the apical and/oraxillary meristems, to trigger floral transitioning of those meristemsand induce flowering, embodiments of the present invention provide arecombinant DNA molecule comprising a FT transgene or coding sequenceoperably linked to a “vegetative stage” promoter to cause, whenintroduced or transformed into a plant, expression of the FT transgeneearlier in the development of the plant (i.e., during the vegetativegrowth phase of the plant) to produce an increased level of FT in targettissues than would otherwise occur in a wild type plant at the samestage of development. Timing FT transgene expression during thevegetative stage(s) of development may be important for affecting one ormore reproductive, flowering and/or yield-related traits or phenotypesby providing a timely inductive signal for the production of anincreased number of floral meristems and successful flowers at one ormore node(s) of the plant. Without being bound by any theory, vegetativestage expression of an FT transgene in a plant may operate tosynchronize and/or increase early flowering at one or more node(s) toproduce more flowers per node of the plant. The promoters describedbelow as a part of the present invention provide options for timing FTexpression.

As used herein, a “vegetative stage” promoter includes any promoter thatinitiates, causes, drives, etc., transcription or expression of itsassociated gene (or transgene) during one or more vegetative stage(s) ofplant development, such as during one or more of Ve, Vc, V1, V2, V3, V4,etc., and/or any or all later vegetative stages of development (e.g., upto V_(n) stage). Such a “vegetative stage” promoter may be furtherdefined as a “vegetative stage preferred” promoter that initiates,causes, drives, etc., transcription or expression of its associated gene(or transgene) at least preferably or mostly, if not exclusively, duringone or more vegetative stage(s) of plant development (as opposed toreproductive stages). However, a “vegetative stage” and a “vegetativestage preferred” promoter may each also permit, allow, cause, etc.,transcription or expression of its associated gene (or transgene) duringreproductive phase(s) of development in one or more cells or tissues ofthe plant. The features and characteristics of these vegetative stagesfor a given plant species are known in the art. For dicot plants,vegetative morphological features and characteristics of the plantduring vegetative stages of development may include cotyledon form,vegetative meristems (apical, lateral/axillary, and root), leafarrangement, leaf shape, leaf margin, leaf venation, petioles, stipules,ochrea, hypocotyl, and roots. According to embodiments of the presentinvention, a “vegetative stage” promoter may also be further defined bythe particular vegetative stage during which observable or pronouncedtranscription or expression of its associated gene (or transgene) isfirst initiated. For example, a vegetative stage promoter may be a Vcstage promoter, a V1 stage promoter, a V2 stage promoter, a V3 stagepromoter, etc. As such, a “Vc stage” promoter is defined as a vegetativestage promoter that first initiates transcription of its associated gene(or transgene) during the Vc stage of plant development, a “V1 stage”promoter is defined as a vegetative stage promoter that first initiatestranscription of its associated gene (or transgene) during the V1 stageof plant development, a “V2 stage” promoter is defined as a vegetativestage promoter that first initiates transcription of its associated gene(or transgene) during the V2 stage of plant development, and so on,although expression of the associated gene (or transgene) may be presentcontinuously or discontinuously in one or more tissues during latervegetative stage(s) of development. One skilled in the art would be ableto determine the timing of expression of a given gene (or transgene)during plant development using various molecular assays and techniquesknown in the art.

According to embodiments of the present invention, a “vegetative stage”promoter may include a constitutive, tissue-preferred, ortissue-specific promoter. For example, a vegetative stage promoter maydrive FT expression in one or more plant tissue(s), such as in one ormore of the root(s), stem(s), leaf/leaves, meristem(s), etc., during avegetative stage(s) of plant development. However, such a vegetativestage promoter may preferably drive expression of its associated FTtransgene or coding sequence in one or more meristem(s) of the plant.According to many embodiments, a “vegetative stage” promoter maypreferably be a “meristem-specific” or “meristem-preferred” promoter tocause expression of the FT transgene or coding sequence in meristematictissue. FT proteins are known to operate in the meristems of a plant tohelp trigger the transition from vegetative to reproductive growth aftertranslocation of the FT protein from the leaves. In contrast,embodiments of the present invention provide selective expression of anFT transgene directly in the meristem of a plant to induce flowering andcause the plant to adopt an altered reproductive and/or yield-relatedtrait or phenotype. Thus, according to embodiments of the presentinvention, a recombinant DNA molecule is provided comprising an FTtransgene or coding sequence operably linked to a “meristem-specific” or“meristem-preferred” promoter that drives expression of the FT transgeneat least preferentially in one or more meristematic tissues of a plantwhen transformed into the plant. As used herein, “meristem-preferredpromoter” refers to promoters that preferentially cause expression of anassociated gene or transgene in at least one meristematic tissue of aplant relative to other plant tissues, whereas a “meristem-specificpromoter” refers to promoters that cause expression of an associatedgene or transgene exclusively (or almost exclusively) in at least onemeristem of a plant.

Recent work using artificial early “short day” light treatments duringvegetative stages of development revealed that flowering time could bealtered in a way that alters one or more yield-related traits orphenotypes (e.g., by causing an increased number of pods or seeds pernode on a plant) and that the effect of these treatments wasdosage-dependent with the number of flowers, seeds and/or pods per plant(and/or per node of the plant) depending on (i) the duration of theshort day exposure (i.e., floral induction signal dosage) and (ii) thelength of the post-short day photoperiods under long day conditions(i.e., the dosage or length of the vegetative growth inducing signalafter the short day induction signal). See, e.g., U.S. Pat. No.8,935,880 and U.S. Patent Application Publication No. 2014/0259905,introduced above. Soybean plants experiencing a lower or less prolongedearly short day induction (eSDI) treatment (prior to returning to longday growing conditions) had more flowers, pods and seeds per plant withmore normal plant height and maturity, whereas soybean plants exposed toa greater or more prolonged eSDI treatment produced shorter,earlier-terminating plants with fewer pods and seeds per plant (albeitperhaps with an increased number of pods and/or seeds per node).

Without being bound by any theory, it is proposed that an earlyflorigenic signal (e.g., short days for soybean and other SD plants)triggers an early vegetative to reproductive transition of plants andeven termination of a subset of its primary meristems. However, byreturning those plants to non-inductive growth conditions (e.g., longdays for SD plants) after the initial SD signal, the remainingmeristematic reserves of the plant may be preserved and reproductiveand/or flowering duration may be extended or maintained, thus allowingfor the successful development of a greater number of productiveflowers, pods and/or seeds per node (and/or per plant) during thereproductive phase. With early floral induction, a greater overlap mayalso be created between reproductive development and vegetative growthof the plant, which may further promote or coincide with an extendedreproductive and/or flowering duration. For purposes of the presentinvention, “reproductive duration” refers to the length of time from theinitiation of flowering until the end of seed/pod development and/orfilling, whereas “flowering duration” or “duration of flowering” refersthe length of time from the appearance of the first open flower untilthe last open flower closes. By returning to non-inductive growthconditions after early floral induction, more abundant resources may beavailable and directed toward the production of an increased number ofsuccessful (i.e., non-aborting) flowers, pods and/or seeds per plant,unlike normal floral development in short day plants, which may coincidewith declining plant resources due to termination of meristematic growthand maturation of the plant.

However, in addition to early flowering, a floral induction signal(e.g., early short day conditions) also causes early termination of theplant. Therefore, it is proposed that an optimal dosage and timing ofthe floral induction signal may be needed to maximize yield by balancing(i) the early vegetative to reproductive transition and/orsynchronization of flowering with the early floral induction signal(leading to potential yield gains at each node of the plant) against(ii) earlier growth termination (leading to smaller plants with fewerinternodes, less branching, and fewer nodes and/or flowers per plant).It is believed that lower dosages of a floral induction signal may besufficient to induce flowering while lessening or minimizing earliertermination of the plant, such that larger plants are produced withincreased numbers of flowers, pods and/or seeds per node (and/or perplant). On the other hand, higher dosages of a floral induction signalmay cause early termination of the plant (in addition to earlyflowering) to produce smaller plants with relatively fewer numbers offlowers, pods and/or seeds per plant due to the smaller plant size withfewer internodes and/or branches per plant, despite having perhaps agreater number of flowers, pods and/or seeds per node (and/or per plant)relative to wild-type or control plants under normal growth conditions.As stated above, these effects of ectopic FT expression may also includean increased number of bolls, siliques, fruits, nuts, tubers, etc., pernode (and/or per plant), depending on the particular plant species.

As mentioned above, the short day induction phenotype in soybean wasused to screen for genes having altered expression in those plantsthrough transcriptional profiling. These studies led to theidentification of an endogenous FT gene, Gm.FT2a, having increasedexpression in response to the short day induction treatment.Accordingly, it is presently proposed that expression of a florigenic FTtransgene can be used as a floral induction signal to cause earlyflowering and increased flowers, pods and/or seeds per node (and/or perplant) relative to a wild type or control plant not having the FTtransgene. According to embodiments of the present invention,appropriate control of the timing, location and dosage of florigenic FTexpression during vegetative stages of development can be used to induceflowering and produce plants having increased flowers, pods and/or seedsper node relative to a wild type or control plant not having the FTtransgene. Rather than attempting to transiently express FT andrecapitulate the timing of the eSDI treatments, it is proposed that FTcould be weakly expressed in the vegetative meristem to provide theearly floral induction signal. Accordingly, a promoter from the Erectagene (pErecta or pEr) having weak meristematic expression duringvegetative stages of development was selected for initial testing with aGm.FT2a transgene. However, given that prior studies showed thatconstitutive FT expression produced plants having a severe, earlytermination phenotype, and further that the site of action for FTproduced peripherally and translocated from the leaves is in themeristem, it was possible that direct meristematic expression of FTcould produce even more potent and severe phenotypes (and/or non-viableplants) relative to constitutive FT expression.

The effects of Gm.FT2a overexpression were immediately seen in R₀transformed soybean plants, which had early flowering, reduced seedyield (e.g., only about 8 seeds/plant), and very early termination,suggesting that the balance between floral induction and floralrepression/vegetative growth was strongly in favor of flowering andearly termination. However, enough R1 seed was salvaged from theseplants to allow for additional experiments to be performed. It wasproposed that growing the Ri soybean seed under long day (floralrepressive) photoperiod conditions in the greenhouse might delay theearly flowering and termination phenotypes observed in the Ro plants.Given the theorized dosage response, it was further proposed thatsegregating FT2a homozygous, hemizygous and null soybean plants could betested together in the greenhouse to evaluate the dosage responseresulting from FT overexpression. In these experiments (as describedfurther below), it was observed that segregating plants did havedifferent phenotypes: null plants were similar to wild-type plants interms of plant architecture and pods per node (and per plant), whilehomozygous plants terminated early with a severe dwarf phenotype(although possibly with an increased number of pods per node). However,hemizygous plants were almost as large as null or wild-type plants butexhibited the hyper-flowering phenotype with an increased number of podsper node (and/or per plant). These findings show that vegetative stageand/or meristematic expression of a florigenic FT transgene may be usedto produce a high yielding plant (similar to the eSDI treatment), andthat the effect of FT expression is dosage-dependent since soybeanplants hemizygous for the FT2a transgene under the control of a weakmeristematic promoter displayed the high yield phenotype of increasedpods per node without the more severe early termination and short plantheight phenotypes observed with homozygous FT2a plants when grown underlong day (vegetative) conditions.

Interestingly, however, increased numbers of pods per node was oftenobserved independently of reproductive (R1-R7) duration under greenhouseconditions and was not perfectly correlated with early flowering amongthe FT transgenes tested, although it is possible that the duration offlowering may still be prolonged in some cases (at least during one ormore reproductive stages) even if the total reproductive duration is notsignificantly changed relative to control plants. Without being bound byany theory, it is believed that increased numbers of pods per node intransgenic FT plants may result at least in part from an increase in thenumber of inflorescence and floral meristems induced from vegetativeshoot apical and axillary meristems at each of the affected node(s),which may give rise to a greater number of flowers and/or releasedfloral racemes at those node(s). Such an increase in the number offloral meristems induced at each node of the plant in response to FToverexpression may operate through one or more mechanisms or pathways,which may be independent of flowering time and/or reproductive duration.However, meristematic changes may be microscopic at first, and thus notobserved to cause “early flowering” at such stage by simple visualinspection even though reproductive changes to the meristem may havealready begun to occur.

Without being bound by any theory, early vegetative FT expression maycause more reproductive meristems to form and develop earlier thannormal at one or more node(s) of the transgenic plant. Thesereproductive meristems may then allow or cause a greater number offloral racemes to form and elongate with flowers at each node. On theother hand, it is further theorized that later expression of FT duringreproductive stages may function to repress further floral developmentat each node. Thus, later developing flowers within the respectiveraceme may become terminated, and thus more of the plant's resources maybe directed to the earlier developing flowers within the raceme to moreeffectively produce full-sized pods. Accordingly, it is contemplatedthat by (i) forming a greater number of inflorescence and floralmeristems at each node by early FT induction, and then (ii) directingmore plant resources to the earlier, more developed flowers within eachof the racemes by termination of the later-developing flowers, increasedsynchronization of floral development may occur with a greater number ofmature pods being formed per node of the plant.

According to embodiments of the present invention, a recombinant DNAmolecule is provided comprising an FT coding sequence operatively linkedto a vegetative stage promoter, which may also be a meristem-preferredand/or meristem-specific promoter. For example, the promoter may includethe pAt.Erecta promoter from Arabidopsis (SEQ ID NO: 21), or afunctional fragment or portion thereof. Two examples of a truncatedportion of the pAt.Erecta promoter according to embodiments of thepresent invention are provided as SEQ ID NO: 22 and SEQ ID NO: 38. See,e.g., Yokoyama, R. et al., “The Arabidopsis ERECTA gene is expressed inthe shoot apical meristem and organ primordia,” The Plant Journal 15(3):301-310 (1998). pAt.Erecta is an example of a vegetative stage promoterthat is also meristem-preferred. Other vegetative stage,meristem-preferred or meristem-specific promoters have been identifiedbased on their characterized expression profile (see, e.g., Examples 4and 7 below) that may also be used to drive FT expression according toembodiments of the present invention. For example, the following soybeanreceptor like kinase (RLK) genes were identified that could be used asvegetative stage, meristem-preferred promoters: Glyma10g38730 (SEQ IDNO: 23), Glyma09g27950 (SEQ ID NO: 24), Glyma06g05900 (SEQ ID NO: 25),and Glyma17g34380 (SEQ ID NO: 26). Vegetative stage, meristem-preferredpromoters according to embodiments of the present invention may alsoinclude receptor like kinase (RLK) gene promoters from potato:PGSC0003DMP400032802 (SEQ ID NO: 27) and PGSC0003DMP400054040 (SEQ IDNO: 28). Given the characterization provided herein of the pAt.Erectapromoter driving FT expression and the similar expression profilesidentified for other RLK, Erecta or Erecta-like (Er1) genes,vegetative-stage, meristem-preferred or meristem-specific promoters ofthe present invention may further comprise any known or later identifiedpromoter sequences of RLK, Erecta and Erecta-like genes from otherdicotyledonous species having vegetative-stage pattern of expression inthe meristems of plants.

Additional examples of vegetative stage, meristem-preferred ormeristem-specific promoters may include those from the followingArabidopsis genes: Pinhead (At.PNH) (SEQ ID NO: 29), Angustifolia 3 orAt.AN3 (SEQ ID NO: 30), At.MYBI7 (At.LMI2 or Late Meristem Identity 2;At3g61250) (SEQ ID NO: 31), Kinesin-like gene (At5g55520) (SEQ ID NO:32), AP2/B3-like genes, including ALREM17 (SEQ ID NO: 33) or ALREA119,and Erecta-like 1 and 2 genes, At.Erl1 (SEQ ID NO: 34) and At.Erl2 (SEQID NO: 35), and functional portions thereof. The polynucleotide sequenceof these promoters (or a functional fragment thereof) may also have arelaxed sequence identity while still maintaining a similar or identicalpattern of expression of an associated gene or transgene operably linkedto the promoter. For example, the promoter may comprise a polynucleotidesequence that is at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or least 99% identical to a polynucleotide sequence selectedfrom the above SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, or 35, or a functional portion thereof. A “functionalportion” of a known or provided promoter sequence is defined as one ormore continuous or discontinuous portion(s) of the known or providedpromoter sequence that may functionally drive, cause, promote, etc.,expression of its associated gene (or transgene) in a manner that isidentical or similar to the known or provided promoter sequence. Basedon the present disclosure, one skilled in the art would be able todetermine if a promoter comprising one or more portion(s) of a known orprovided promoter sequence, and/or having a shorter sequence and/or morerelaxed sequence identity relative to a known or provided promotersequence, causes a similar pattern of expression and/or similarphenotypes or effects when its associated FT transgene is expressed in aplant as compared to the known or provided promoter sequence.

As stated above, a recombinant DNA molecule of the present invention maygenerally comprise an FT transgene or expression cassette including apolynucleotide sequence encoding an FT protein that is operativelylinked to a vegetative stage promoter, which may also be ameristem-preferred or meristem-specific promoter. The polynucleotidecoding sequence of the FT transgene or expression cassette may also beoperatively linked to one or more additional regulatory element(s), suchas an enhancer(s), leader, transcription start site (TSS), linker, 5′and 3′ untranslated region(s), intron(s), polyadenylation signal,termination region or sequence, etc., that are suitable or necessary forregulating or allowing expression of the FT transgene or cassette toeffectively produce an FT protein in a plant cell. Such additionalregulatory element(s) may be optional and used to enhance or optimizeexpression of the transgene. For purposes of the present invention, an“enhancer” may be distinguished from a “promoter” in that an enhancertypically lacks a transcription start site, TATA box, or equivalentsequence and is thus insufficient alone to drive transcription. As usedherein, a “leader” may be defined generally as the DNA sequence of theuntranslated 5′ region (5′ UTR) of a gene (or transgene) between thetranscription start site (TSS) and the protein coding sequence startsite.

According to embodiments of the present invention, the term“recombinant” in reference to a DNA molecule, construct, vector, etc.,refers to a DNA molecule or sequence that is not found in nature and/oris present in a context in which it is not found in nature, including aDNA molecule, construct, etc., comprising a combination of DNA sequencesthat would not naturally occur contiguously or in close proximitytogether without human intervention, and/or a DNA molecule, construct,etc., comprising at least two DNA sequences that are heterologous withrespect to each other. A recombinant DNA molecule, construct, etc., maycomprise DNA sequence(s) that is/are separated from other polynucleotidesequence(s) that exist in proximity to such DNA sequence(s) in nature,and/or a DNA sequence that is adjacent to (or contiguous with) otherpolynucleotide sequence(s) that are not naturally in proximity with eachother. A recombinant DNA molecule, construct, etc., may also refer to aDNA molecule or sequence that has been genetically engineered andconstructed outside of a cell. For example, a recombinant DNA moleculemay comprise any suitable plasmid, vector, etc., and may include alinear or circular DNA molecule. Such plasmids, vectors, etc., maycontain various maintenance elements including a prokaryotic origin ofreplication and selectable marker, as well as a FT expressing transgeneor expression cassette perhaps in addition to a plant selectable markergene, etc.

As introduced above, the florigenic effects of FT expression may beopposed by the activity of various other anti-florigenic (ornon-florigenic) proteins, such as Terminal Flower (TFL) genes, in themeristem of a plant. Flowering time and duration may thus be seen as abalance between florigenic and anti-florigenic signals present withinthe meristem(s) of a plant. Accordingly, it is further proposed thatflowering time in a plant may be altered or induced by suppression ofone or more anti-florigenic genes during vegetative stages ofdevelopment to render the vegetative meristem more responsive to aflorigenic signal. In soybean, for example, genes related to ArabidopsisTFL1 are primarily expressed in floral meristems where they oppose thefunctions of florigenic signals to regulate developmental decisions likestem growth habit. In particular, the TFL1-like genes, TFL1a and TFL1b,in soybean are expressed in the shoot apical meristem, and allelicdiversity in TFL1b may be largely responsible for changes in stem growthhabit in soy resulting in determinate or indeterminate growth. See,e.g., Liu, B. et al., “The soybean stem growth habit gene Dt1 is anortholog of Arabidopsis TERMINAL FLOWER 1.” Plant Physiol 153(1):198-210(2010). Accordingly, suppressing the expression of TFL or anotheranti-florigenic protein in the meristem of a dicot plant may result inincreased sensitivity of those meristematic tissues to florigenicsignals, such as FT, resulting in early flowering and increased pods pernode, similarly to overexpression of FT in these tissues.

Thus, it is contemplated that a recombinant DNA molecule of the presentinvention may further comprise a suppression construct having atranscribable DNA sequence operatively linked to a vegetative stagepromoter, which may also be a meristem-preferred or meristem-specificpromoter, wherein the transcribable DNA sequence encodes an RNA moleculethat causes targeted suppression of an endogenous anti-florigenic gene,such as a TFL gene. Various methods for suppressing the expression of anendogenous gene are known in the art.

According to another broad aspect of the present invention, methods areprovided for transforming a plant cell, tissue or explant with arecombinant DNA molecule or construct comprising an FT transgene orexpression cassette to produce a transgenic plant. Numerous methods fortransforming chromosomes in a plant cell with a recombinant DNA moleculeare known in the art, which may be used according to methods of thepresent invention to produce a transgenic plant cell and plant. Anysuitable method or technique for transformation of a plant cell known inthe art may be used according to present methods. Effective methods fortransformation of plants include bacterially mediated transformation,such as Agrobacterium-mediated or Rhizhobium-mediated transformation,and microprojectile bombardment-mediated transformation. A variety ofmethods are known in the art for transforming explants with atransformation vector via bacterially mediated transformation ormicroprojectile bombardment and then subsequently culturing, etc, thoseexplants to regenerate or develop transgenic plants. Other methods forplant transformation, such as microinjection, electroporation, vacuuminfiltration, pressure, sonication, silicon carbide fiber agitation,PEG-mediated transformation, etc., are also known in the art. Transgenicplants produced by these transformation methods may be chimeric ornon-chimeric for the transformation event depending on the methods andexplants used. Methods are further provided for expressing an FTtransgene in one or more plant cells or tissues under the control of avegetative-stage promoter, which may also be a meristem-preferred ormeristem-specific promoter. Such methods may be used to alter floweringtime of a plant and/or the number of productive or successful flowers,fruits, pods, and/or seeds per node of the plant relative to a wild typeor control plant not having the FT transgene. Indeed, methods of thepresent invention may be used to alter reproductive or yield-relatedphenotype(s) or trait(s) of the transgenic plant.

Transformation of a target plant material or explant may be practiced intissue culture on nutrient media, for example a mixture of nutrientsthat allow cells to grow in vitro. Recipient cell targets or explantsmay include, but are not limited to, meristems, shoot tips, protoplasts,hypocotyls, calli, immature or mature embryos, shoots, buds, nodalsections, leaves, gametic cells such as microspores, pollen, sperm andegg cells, etc., or any suitable portions thereof. It is contemplatedthat any transformable cell or tissue from which a fertile plant can beregenerated or grown/developed may be used as a target fortransformation. Transformed explants, cells or tissues may be subjectedto additional culturing steps, such as callus induction, selection,regeneration, etc., as known in the art. Transformed cells, tissues orexplants containing a recombinant DNA insertion may be grown, developedor regenerated into transgenic plants in culture, plugs or soilaccording to methods known in the art. Transgenic plants may be furthercrossed to themselves or other plants to produce transgenic seeds andprogeny. A transgenic plant may also be prepared by crossing a firstplant comprising the recombinant DNA sequence or transformation eventwith a second plant lacking the insertion. For example, a recombinantDNA sequence may be introduced into a first plant line that is amenableto transformation, which may then be crossed with a second plant line tointrogress the recombinant DNA sequence into the second plant line.Progeny of these crosses can be further back crossed into the moredesirable line multiple times, such as through 6 to 8 generations orback crosses, to produce a progeny plant with substantially the samegenotype as the original parental line but for the introduction of therecombinant DNA sequence.

A recombinant DNA molecule or construct of the present invention may beincluded within a DNA transformation vector for use in transformation ofa target plant cell, tissue or explant. Such a transformation vector ofthe present invention may generally comprise sequences or elementsnecessary or beneficial for effective transformation in addition to theFT expressing transgene or expression cassette. ForAgrobacterium-mediated transformation, the transformation vector maycomprise an engineered transfer DNA (or T-DNA) segment or region havingtwo border sequences, a left border (LB) and a right border (RB),flanking at least the FT expressing transgene or expression cassette,such that insertion of the T-DNA into the plant genome will create atransformation event for the FT transgene or expression cassette. Inother words, the FT transgene or expression cassette would be locatedbetween the left and right borders of the T-DNA, perhaps along with anadditional transgene(s) or expression cassette(s), such as a plantselectable marker transgene and/or other gene(s) of agronomic interestthat may confer a trait or phenotype of agronomic interest to a plant.In addition to protein encoding sequences, a gene of agronomic interestmay further comprise a polynucleotide sequence encoding a RNAsuppression element. According to alternative embodiments, the FTtransgene or expression cassette and the plant selectable markertransgene (or other gene of agronomic interest) may be present inseparate T-DNA segments on the same or different recombinant DNAmolecule(s), such as for co-transformation. A transformation vector orconstruct may further comprise prokaryotic maintenance elements, whichfor Agrobacterium-mediated transformation may be located in the vectorbackbone outside of the T-DNA region(s).

A plant selectable marker transgene in a transformation vector orconstruct of the present invention may be used to assist in theselection of transformed cells or tissue due to the presence of aselection agent, such as an antibiotic or herbicide, wherein the plantselectable marker transgene provides tolerance or resistance to theselection agent. Thus, the selection agent may bias or favor thesurvival, development, growth, proliferation, etc., of transformed cellsexpressing the plant selectable marker gene, such as to increase theproportion of transformed cells or tissues in the Ro plant. Commonlyused plant selectable marker genes include, for example, thoseconferring tolerance or resistance to antibiotics, such as kanamycin andparomomycin (nptll), hygromycin B (aph IV), streptomycin orspectinomycin (aadA) and gentamycin (aac3 and aacC4), or thoseconferring tolerance or resistance to herbicides such as glufosinate(bar or pat), dicamba (DMO) and glyphosate (aroA or EPSPS). Plantscreenable marker genes may also be used, which provide an ability tovisually screen for transformants, such as luciferase or greenfluorescent protein (GFP), or a gene expressing a beta glucuronidase oruidA gene (GUS) for which various chromogenic substrates are known.

According to embodiments of the present invention, methods fortransforming a plant cell, tissue or explant with a recombinant DNAmolecule or construct may further include site-directed or targetedintegration. According to these methods, a portion of a recombinant DNAdonor template molecule (i.e., an insertion sequence) may be inserted orintegrated at a desired site or locus within the plant genome. Theinsertion sequence of the donor template may comprise a transgene orconstruct, such as an FT transgene or construct comprising apolynucleotide sequence encoding a florigenic FT protein operativelylinked to a vegetative-stage promoter, which may also be ameristem-preferred or meristem-specific promoter. The donor template mayalso have one or two homology arms flanking the insertion sequence topromote the targeted insertion event through homologous recombinationand/or homology-directed repair. Thus, a recombinant DNA molecule of thepresent invention may further include a donor template for site-directedor targeted integration of a transgene or construct, such as an FTtransgene or construct, into the genome of a plant.

Any site or locus within the genome of a plant may potentially be chosenfor site-directed integration of a transgene or construct of the presentinvention. For site-directed integration, a double-strand break (DSB) ornick may first be made at a selected genomic locus with a site-specificnuclease, such as, for example, a zinc-finger nuclease, an engineered ornative meganuclease, a TALE-endonuclease, or an RNA-guided endonuclease(e.g., Cas9 or Cpf1). Any method known in the art for site-directedintegration may be used. In the presence of a donor template molecule,the DSB or nick may then be repaired by homologous recombination betweenthe homology arm(s) of the donor template and the plant genome, or bynon-homologous end joining (NHEJ), resulting in site-directedintegration of the insertion sequence into the plant genome to createthe targeted insertion event at the site of the DSB or nick. Thus,site-specific insertion or integration of a transgene or construct maybe achieved.

According to embodiments of the present invention, a plant that may betransformed with a recombinant DNA molecule or transformation vectorcomprising an FT transgene may include a variety of flowering plants orangiosperms, which may be further defined as including variousdicotyledonous (dicot) plant species, such as soybean, cotton, alfalfa,canola, sugar beets, alfalfa and other leguminous plants. A dicot plantcould be a member of the Brassica sp. (e.g., B. napus, B. rapa, B.juncea), particularly those Brassica species useful as sources of seedoil, alfalfa (Medicago sativa), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), oil palm (Elaeis spp.), sesame (Sesamum spp.),coconut (Cocos spp.), soybean (Glycine max), tobacco (Nicotianatabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Coffea spp.), tea(Camellia spp.), fruit trees, such as apple (Mahis spp.), Prunus spp.,such as plum, apricot, peach, cherry, etc., pear (Pyrus spp.), fig(Ficus casica), banana (Musa spp.), etc., citrus trees (Citrus spp.),cocoa (Theobroma cacao), avocado (Persea americana), olive (Oleaeuropaea), almond (Prunus amygdalus), walnut (Juglans spp.), strawberry(Fragaria spp.), watermelon (Citrullus lanatus), pepper (Capsicum spp.),sugar beet (Beta vulgaris), grape (Vitis, Muscadinia), tomato(Lycopersicon esculentum, Solanum lycopersicum), and cucumber (Cucumissativis). Leguminous plants include beans and peas. Beans include, forexample, guar, locust bean, fenugreek, soybean, garden beans, cowpea,mungbean, lima bean, fava bean, lentils, and chickpea. Given that thepresent invention may apply to a broad range of plant species, thepresent invention further applies to other botanical structuresanalogous to pods of leguminous plants, such as bolls, siliques, fruits,nuts, tubers, etc. According to embodiments of the present invention anddepending on the particular plant species transformed, a plantectopically expressing a florigenic FT sequence may have an altered orgreater number of bolls, siliques, fruits, nuts, tubers, etc., pernode(s), main stem, and/or branch(es) of the plant, and/or an altered orgreater number of bolls, siliques, fruits, nuts, tubers, etc., perplant, relative to a wild type or control plant not having the FTtransgene.

According to another broad aspect of the present invention, a transgenicplant(s), plant cell(s), seed(s), and plant part(s) are providedcomprising a transformation event or insertion into the genome of atleast one plant cell thereof, the transformation event or insertioncomprising a recombinant DNA sequence, construct or polynucleotideincluding a Flowering Locus T (FT) transgene or expression cassette,wherein the FT transgene or expression cassette further comprises apolynucleotide sequence encoding an FT protein operably linked to avegetative stage promoter, which may also be a meristem-preferred ormeristem-specific promoter. The FT protein encoded by the polynucleotidesequence may be native to the transgenic plant transformed with thepolynucleotide sequence, or homologous or otherwise similar to a FTprotein native to the transgenic plant (i.e., not native to thetransgenic plant). Such a transgenic plant may be produced by anysuitable transformation method, which may be followed by selection,culturing, regeneration, development, etc., as desired or needed toproduce a transgenic Ro plant, which may then be selfed or crossed toother plants to generate R1 seed and subsequent progeny generations andseed through additional crosses, etc. Similarly, embodiments of thepresent invention further include a plant cell, tissue, explant, etc.,comprising one or more transgenic cells having a transformation event orgenomic insertion of a recombinant DNA or polynucleotide sequencecomprising an FT transgene.

Transgenic plants, plant cells, seeds, and plant parts of the presentinvention may be homozygous or hemizygous for a transgenic event orinsertion of an FT transgene or expression cassette into the genome ofat least one plant cell thereof, or may contain any number of copies ofa transgenic event(s) or insertion(s) comprising an FT transgene orexpression cassette. The dosage or amount of expression of an FTtransgene or expression cassette may be altered by its zygosity and/ornumber of copies, which may affect the degree or extent of phenotypicchanges in the transgenic plant, etc. According to some embodiments, atransgenic plant comprising an FT transgene may be further characterizedas having one or more altered flowering or reproductive phenotypes ortraits, which may include altered yield-related phenotypes or traits,such as an increase in the number of flowers, pods, etc., and/or seedsper plant (and/or per node of the plant) relative to a wild type orcontrol plant not having the FT transgene. Such a transgenic plant maybe further characterized as having an altered structure, morphology,and/or architecture due to altered plant height, branching patterns,number of floral nodes, etc., relative to a wild type or control plant.Indeed, yield-related phenotypes or traits altered by FT overexpressionin a transgenic plant may include: flowering time, reproductiveduration, flowering duration, amount or timing of abscission of flowers,pods, siliques, bolls, fruits, nuts, etc., number of flowers per node,number of racemes per node, number of branches, number of nodes perplant, number of nodes on the main stem, number of nodes on branches,number of pods, bolls, siliques, fruits, nuts, etc., per plant, numberof pods, bolls, siliques, fruits, nuts, etc., per node, number of podson the main stem, number of pods, bolls, siliques, fruits, nuts, etc.,on branches, 1000 seed weight, number of seeds per plant, number ofseeds per node, and/or altered plant architecture, as compared to a wildtype or control plant not having the FT transgene.

For purposes of the present invention, a “plant” may include an explant,seedling, plantlet or whole plant at any stage of regeneration ordevelopment. As used herein, a “transgenic plant” refers to a plantwhose genome has been altered by the integration or insertion of arecombinant DNA molecule, construct or sequence. A transgenic plantincludes an Ro plant developed or regenerated from an originallytransformed plant cell(s) as well as progeny transgenic plants in latergenerations or crosses from the Ro transgenic plant. As used herein, a“plant part” may refer to any organ or intact tissue of a plant, such asa meristem, shoot organ/structure (e.g., leaf, stem and tuber), root,flower or floral organ/structure (e.g., bract, sepal, petal, stamen,carpel, anther and ovule), seed (e.g., embryo, endosperm, and seedcoat), fruit (e.g., the mature ovary), propagule, or other plant tissues(e.g., vascular tissue, ground tissue, and the like), or any portionthereof. Plant parts of the present invention may be viable, nonviable,regenerable, and/or non-regenerable. A “propagule” may include any plantpart that is capable of growing into an entire plant. For purposes ofthe present invention, a plant cell transformed with an FT transgeneaccording to embodiments of the present invention may include any plantcell that is competent for transformation as understood in the art basedon the method of transformation, such as a meristem cell, an embryoniccell, a callus cell, etc. As used herein, a “transgenic plant cell”simply refers to any plant cell that is transformed with astably-integrated recombinant DNA molecule or sequence. A transgenicplant cell may include an originally-transformed plant cell, atransgenic plant cell of a regenerated or developed Ro plant, or atransgenic plant cell from any progeny plant or offspring of thetransformed Ro plant, including cell(s) of a plant seed or embryo, or acultured plant or callus cell, etc.

Embodiments of the present invention may further include methods formaking or producing transgenic plants having altered reproductive and/oryield-related traits or phenotypes, such as by transformation, crossing,etc., wherein the method comprises introducing a recombinant DNAmolecule or sequence comprising an FT transgene into a plant cell, andthen regenerating or developing the transgenic plant from thetransformed plant cell, which may be performed under selection pressurefavoring the transgenic event. Such methods may comprise transforming aplant cell with a recombinant DNA molecule or sequence comprising an FTtransgene, and selecting for a plant having one or more alteredphenotypes or traits, such as one or more of the following: floweringtime, reproductive duration, flowering duration, amount or timing ofabscission of flowers, pods, bolls, siliques, fruits, nuts, etc., numberof flowers per node, number of racemes per node, number of branches,number of nodes per plant, number of nodes on the main stem, number ofnodes on branches, number of pods, bolls, siliques, fruits, or nuts perplant, number of pods, bolls, siliques, fruits, nuts, etc., per node,number of pods, bolls, siliques, fruits, nuts, etc., on the main stem,number of pods, bolls, siliques, fruits, nuts, etc., on branches, 1000seed weight, number of seeds per plant, number of seeds per node, andaltered plant architecture, as compared to a wild type or control plantnot having the FT transgene. For example, embodiments of the presentinvention may comprise methods for producing a transgenic plant havingan increased number of flowers, pods, and/or seeds per plant (and/or anincreased number of flowers, pods, and/or seeds per node of the plant),wherein the method comprises introducing a recombinant DNA moleculecomprising an FT transgene into a plant cell, and then regenerating ordeveloping the transgenic plant from the plant cell.

According to another broad aspect of the present invention, methods areprovided for planting a transgenic plant(s) of the present invention ata normal or high density in field. According to some embodiments, theyield of a crop plant per acre (or per land area) may be increased byplanting a transgenic plant(s) of the present invention at a higherdensity in the field. As described herein, transgenic plants of thepresent invention expressing a florigenic FT protein during vegetativestage(s) of development may exhibit increased pods per node(particularly on the main stem), but may also have an altered plantarchitecture with reduced branching and fewer nodes per branch. Thus, itis proposed that transgenic plants of the present invention may beplanted at a higher density to increase yield per acre in the field. Forrow crops, higher density may be achieved by planting a greater numberof seeds/plants per row length and/or by decreasing the spacing betweenrows. According to some embodiments, a transgenic crop plant of thepresent invention may be planted at a density in the field (plants perland/field area) that is at least 5%, 10%, 15%, 20%, 25%, 50%, 75%,100%, 125%, 150%, 175%, 200%, 225%, or 250% higher than a normalplanting density for that crop plant according to standard agronomicpractices.

For soybean, the typical planting density is in a range from about100,000 to 150,000 seeds per acre, and the typical row spacing is in arange from about 26 to about 40 inches, such as 30 inch or 36 inch rowspacing. Within a given row, about 6-8 soybean seeds may typically beplanted per foot. In contrast, high density planting for soybean mayinclude a range of approximately 150,000 to 250,000 seeds per acre, andthe row spacing may be within a range from about 10 inches or less toabout 25 inches, such as 10 inch, 15 inch or 20 inch row spacing. Forhigh density planting, approximately 9-12 soybean seeds per foot may beplanted within each row, perhaps in combination with narrower rowspacing. However, high crop density may be achieved by narrow rowspacing without an increase in planting density within each row.

For cotton, the typical planting density is in a range from about 28,000to 45,000 seeds per acre, and the typical row spacing is in a range fromabout 38 to about 40 inches, such as 38 inch or 40 inch row spacing.Within a given row, about 2-3 cotton seeds may typically be planted perfoot. In contrast, high density planting for soybean may include a rangeof approximately 48,000 to 60,000 seeds per acre, and the row spacingmay be within a range from about 30 inches or less to about 36 inches.For high density planting, approximately 3-5 cotton seeds per foot maybe planted within each row, perhaps in combination with narrower rowspacing. However, high crop density for cotton may be achieved by narrowrow spacing without an increase in planting density within each row.

For canola, the typical planting density is in a range from about360,000 to 550,000 seeds per acre, and the typical row spacing betweenopeners is in a range from about 6 inches to about 16 inches. Within agiven row, about 8-12 canola seeds may typically be planted per foot. Incontrast, high density planting for soybean may include a range ofapproximately 450,000 to 680,000 seeds per acre, and the row spacing maybe within a range from about 5 inches or less to about 10 inches. Forhigh density planting, approximately 10-16 canola seeds per foot may beplanted within each row, perhaps in combination with the narrower rowspacing. However, high crop density for canola may be achieved by narrowrow spacing without an increase in planting density within each row.

EXAMPLES Example 1 Soybean Short Day Induction Treatment andIdentification of Flowering Locus T (FT) Genes by TranscriptionalProfiling

Methods for the photoperiodic light treatment (i.e., short day inductionof flowering in plants) are described in U.S. Pat. No. 8,935,880 andU.S. Patent Application Publication No. 2014/0259905, which areincorporated herein by reference in their entirety. As described furthertherein, the early short day induction treatment produced soybean plantshaving altered reproductive traits including an increased number ofpods/seeds per plant. Transcriptional profiling experiments wereperformed using gene expression microarrays to determine if particulartranscripts were up-regulated in these light-induced plants to identifygenes that may be responsible for mediating the short day inductionphenotypes. In these experiments, an analysis of transcripts wasconducted on soybean leaf and floral apex tissues collected after 1, 3and 5 days from plants that received a short day inductive lighttreatment (Short day) in comparison to tissues from plants that did notreceive the inductive treatment (Long day).

As shown in FIG. 2, an increased accumulation of transcripts wasobserved for a particular Flowering Locus T gene, Gm.FT2a (SEQ ID NO:1), in leaf tissue harvested at 3 and 5 days after the early short dayinduction (eSDI) treatment in comparison to samples taken from either(i) floral apex tissues of the same short day induction plants, or (ii)leaf tissues and floral apex tissues of soybean plants that insteadreceived the long day treatment. These data support the conclusion thatGm.FT2a expression is induced in leaf tissue of plants experiencing theeSDI treatment, which was not seen in plants grown under long dayconditions. Gm.FT2a expression was also not observed in the floral apexof eSDI treated plants, which is consistent with the model of FT proteinexpression being induced in peripheral leafy tissues in response toinductive photoperiod conditions and then migrating to its site ofaction in the meristems to induce flowering. However, additionalexperiments using a more sensitive RNA sequencing analysis oftranscripts did show some Gm.FT2a induction in the shoot apex andaxillary buds in response to the eSDI treatment (data not shown).

Example 2 Characterization of the pAtErecta Promoter Expression Patternsin Soybean

Achieving desirable traits or phenotypes by transgenic approaches mayrequire control of the temporal and spatial patterns of ectopic FT geneexpression. Soy physiological experiments identifying Gm.FT2a expressionin vegetative tissues following the short day induction treatment (seeFIG. 2) indicated that achieving yield positive traits may rely onearlier FT expression during the vegetative stage. On the other hand,even though FT transcripts are not detected in the vegetative apex, FTprotein has been shown to move long distance from the leaves to theapical tissue where it triggers a vegetative to reproductive transition.See, e.g., Lifschitz, E. et al., (2006), supra; and Corbeiser, L. etal., “FT Protein Movement Contributes to Long-Distance Signaling inFloral Induction of Arabidopsis”, Science 316: 1030-1033 (2007). Thus,in light of our own observations, we proposed using a vegetative stagepromoter that is active in the meristem to control ectopic FT expressionin a plant. By expressing the morphogenetic FT signal directly in themeristem at the desired developmental stage, multiple endogenouspathways and regulatory feedbacks (e.g., control of FT transduction inthe leaf and long distance translocation of the FT signal) may bebypassed or avoided. Previous experiments with the short day inductiontreatment (described above in Example 1) revealed up-regulation of theGm.Erecta gene in the meristems of soybean plants. The pErecta promoter(SEQ ID NO: 21) from Arabidopsis had been shown to have weak expressionin the meristem(s) of plants during vegetative stages of development.Accordingly, the pAt.Erecta promoter was selected for initial FTexpression experiments.

Additional experiments were performed to further characterize theexpression patterns of pAt.Erecta fused to a GUS reporter gene invegetative and floral meristematic tissues. Analysis of GUS expressionpatterns during the development of soy seedlings indicated that thepAt.Erecta promoter exhibits a temporal and spatial pattern ofexpression, preferably in the meristematic tissues during the vegetativestage of development. FIGS. 3A to 3O and 4A to 4O and FIGS. 5A to 5F and6A to 6F include two sets of images to show the pattern of GUS staining.FIGS. 3A to 3O and 5A to 5F provide black and white images of thestained tissues, and FIGS. 4A to 4O and 6A to 6F provide black and whiteimages corresponding to FIGS. 3A to 3O and 5A to 5F, respectively, butcolor filtered to show the pattern and intensity of blue GUS staining.Thus, the GUS staining pattern of expression can be viewed with theseblack and white images by comparing the corresponding images of FIGS. 3Ato 3O and 4A to 4O or FIGS. 5A to 5F and 6A to 6F. As shown in FIGS. 3Ato 3O and 4A to 4O, GUS staining was detected in the soy immatureuni-foliate blade and petiole (FIGS. 4A and 4B) at three days aftersowing/germination. pAt.Erecta:GUS expression was also broadly detectedin the trifoliate primordia, shoot apical meristem (SAM) and axillarymeristem sites at this early vegetative stage (FIG. 4C). GUS activitywas not detected in the fully expanded uni-foliate and trifoliate leavesat ten days after germination or planting (FIGS. 4D and 4E). However,GUS activity was detected at the proximal part of the immature,unexpanded, but fully developed trifoliate blade, and at the adaxialside of the petiole (FIG. 4F). Detailed observation of the developingapical tissue showed that broad expression was retained in thedeveloping immature leaf primordia, axillary meristems and shoot apicalmeristems (FIGS. 4G-I).

At the early reproductive stage, pAt.Erecta promoter activity was notdetected in the mature blade and was reduced in the developing leafprimordia. The GUS signal was not detected in the indeterminatevegetative apex at the shoot apical meristem (SAM) or in the axillarymeristem (AM) once these tissues started to form inflorescences (FIGS.4J-4L). In all later stages, any additional meristematic activity couldnot be detected in the apex or in the axillaries or flower primordia.However, GUS expression continued in the adaxial side of the petiole andproximal part of the immature leaf blade (FIGS. 4M and 4N), but not inthe fully expanded leaf blade (FIG. 4O). GUS expression patterns withthe pAt.Erecta promoter were also analyzed at the later R1 stages ofdevelopment (35-40 days after germination). Similar to earlier stages ofdevelopment, no expression was detected in the mature leaves or stems.However, strong promoter activity was detected in the inflorescencestems (FIGS. 5A and 6A; see arrow) and floral pedicels (FIGS. 5B and 6B;see arrow). In both tissues, expression was detected in vasculature andparenchyma cells (FIGS. 5C and 6C). At this stage, expression was alsodetected in the stamen filaments (FIG. 5D and 6D; see arrows) and in theun-pollinated ovules (FIGS. 5E, 5F, 6E and 6F; see arrows in 6F).

Previously, the pAt.Erecta promoter was characterized in Arabidopsis.Interestingly, pAt.Erecta expression patterns in Arabidopsis werecomparable to the patterns observed in soy during the vegetative stage,but not during late reproductive stages. In contrast, the pAt.Erectaexpression pattern in soybean is diminished in early reproductivetissues but remerges in some later reproductive organs and tissues,including the inflorescence stems and floral pedicels. See, e.g., Chen,M-K et al., FEBS Letters 588: 3912-17 (2014); Yokoyama, R et al.; Shpak,E D et al., Science 309: 290-293 (2005); and Yokoyama, Ret al., Plant J15(3): 301-310 (1998), the entire contents and disclosures of which areincorporated herein by reference. Thus, the pAt.Erecta promoter providesa novel expression pattern in soybean.

Example 3 Expression of Flowering Locus T Gene, Gm.FT2a, Under Controlof a pAt Erecta Promoter Alters Flowering Time and Pods Per Node inSoybean

Transgenic soybean plants were produced by transforming soybean explantswith a recombinant DNA molecule (i.e., a T-DNA transformation vector)comprising the pAt.Erecta promoter operably linked to the Gm.FT2a genevia Agrobacterium-mediated transformation to generate fourpErecta::Gm.FT2a events that were carried forward for further testing.The effect of FT2a overexpression was immediately seen in R₀ plants,which had very early flowering and termination with reduced seed yield(e.g., only about 8 seeds/plant). These transgenic Gm.FT2a plants alsohad a short plant height and very few, if any, branches. Segregating R1plants and their progeny were subsequently grown in the greenhouse underlong day conditions for initial study and characterization. By growingthese plants under long day conditions, the severe dwarf phenotypesobserved with Gm.FT2a transgenic Ro plants were improved. In theseexperiments, both homozygous and hemizygous plants grown in thegreenhouse under 16-hour long day conditions (i.e., 16/8 hours ofday/night photoperiods) flowered much earlier than wild type nullsegregants. Gm.FT2a transgenic plants flowered at about 19-22 days afterplanting or seeding). (see, e.g., FIGS. 9A to 9C). Under these growthconditions, transgenic soybean plants expressing Gm.FT2a further had anincreased number of pods per node on the main stem in comparison to wildtype controls (see, e.g., FIGS. 10 and 11, discussed further below).

Plants containing one of the pEr::Gm.FT2a transgenic events (Event 1)grown in controlled environment conditions were further analyzed viascanning electron microscopy analysis (eSEM). Analysis of the shootapical meristem (SAM) of these transgenic plants (collected at 7 daysafter planting) revealed an early transition of the SAM into aninflorescence meristem (IM) and floral meristem (FM) (FIG. 7). Incontrast, the SAMs of wild type soybean plants were not differentiatedinto IM at this growth stage. Similarly, imaging of the axillarymeristem of the FT2a transformants (collected at 9 days after planting)indicated the development of dormant inflorescence meristems (dIMs) (orlateral primordial racemes) into IM and FM (FIG. 8), leading to moreearlier-developing floral branches (racemes) per node in thesetransgenic plants. Additional phenotypic characterization revealed earlyflowering at the V1 stage in Gm.FT2a expressing soybean plants, whichwas well before the floral transition occurred in null segregatingplants (FIGS. 9A to 9C). These data in combination with thepAt.Erecta:GUS expression pattern described above indicate that earlyflowering, and more particularly the formation of inflorescence andfloral meristems, were induced by ectopic expression of Gm.FT2a duringthe vegetative stage in leaf primordia and the shoot apical and axillarymeristems of seedlings. The formation of a higher number ofinflorescence and floral meristems is believed to further cause earlierrelease and elongation of the secondary and tertiary racemes, leading toa greater number of productive flowers and pods being formed per node.

Not only did Gm.FT2a transgenic soybean plants experience earlierflowering and produce more pods per node on the main stem (relative tosegregating null plants), the effects of ectopic Gm.FT2a expression intransgenic plants were also found to be dosage dependent. Although bothhomozygous and hemizygous plants had a reduced height and lessbranching, plants homozygous for the Gm.FT2a transgene were moreseverely affected than hemizygous plants, presumably because homozygousplants contain two copies of the transgene (i.e., a higher dosage), asopposed to only one copy (i.e., a lower dosage) in hemizygous plants.Under long day growth conditions, homozygous plants terminated earlierand had a shorter overall height with fewer nodes and branches on themain stem in comparison to plants hemizygous for the transgene (FIG.10). Unlike homozygous plants, which exhibited a number of sub-optimaldwarf phenotypes including very few (if any) branches on the main stem,hemizygous plants had an intermediate phenotype in terms of theirvegetative growth, plant height, and the number of nodes present on themain stem relative to wild type and homozygous plants. Under 16-hourlong day conditions, hemizygous plants had a more normal plant heightwith some degree of branching and a more extended duration of flowering,relative to homozygous plants (FIG. 10). Hemizygous plants also floweredfor 40-64 days after initiation of R1, whereas homozygous plantsflowered for only 16-24 days due to their earlier termination.

Additional experiments were conducted with plants transformed with theGm.FT2a construct (3 events) in long day (16 hour) controlledenvironment conditions to further characterize the dosage responsebetween hemizygous and homozygous plants. Differences in the number ofnodes and pods on the main stem and branches, as well as the averagenumber of pods per node and the average height per plant are shown inTable 1 for three homozygous events (Homo-Event 2, Homo-Event 3,Homo-Event 4) and three hemizygous events (Hemi-Event 2, Hemi-Event 3,Hemi-Event 4). These events are distinguished from Event 1 above.

TABLE 1 Event level data for homozygous and hemizygous Gm.FT2atransgenic plants. Avg. # MS Avg. # BR Avg. # MS Avg. # BR Avg.Zygosity- nodes per nodes pods pods Avg. Pods Height (in) Event # plantper plant per plant per plant per Node per plant Homo- 11.8 6.9 46 9 2.917.5 Event 2 Homo- 12.3 6.5 66.4 9 4 21 Event 3 Homo- 12.5 6.8 49.6 9.13 19.5 Event 4 Hemi- 25.3 12.4 183.5 47.3 6.1 37.5 Event 2 Hemi- 23.913.2 200.3 28.8 6.1 40 Event 3 Hemi- 25.4 15.3 186.8 58 6 41.5 Event 4

As shown in Table 1, hemizygous plants consistently had a higher numberof nodes on the main stem (MS) and branches (BR) and a greater plantheight than homozygous plants. Thus, hemizygous plants were generallyless affected than homozygous plants and more like wild type plants.Hemizygous plants also had an increased number of pods per node and ahigher number of pods on the main stem and branches, relative tohomozygous plants. Therefore, hemizygous plants generally had acloser-to-normal plant architecture with a greater number of pods pernode (and per plant), presumably due to their lower Gm.FT2a transgenedosage. The relative dosage level of Gm.FT2a based on transgene zygositywas further confirmed by additional experiments showing that Gm.FT2atranscript levels were higher in tissues from homozygous plants, than intissues from hemizygous plants (data not shown).

The early induction of flowering in these Gm.FT2a transgenic plants wasassociated with more pods (and seeds) per node on the main stem in bothhemizygous and homozygous plants. Homozygous and hemizygous plantscontaining the Gm.FT2a transgene each had an increased number ofpods/seeds per node on the main stem of the plant in comparison to wildtype segregants (FIG. 11). The distribution of pods on the main stem wasalso found to be different between FT2a transgenic and wild type nullplants. Both homozygous and hemizygous plants grown under long dayconditions were found to have more pods on at least the lower nodes ofthe main stem and more pods per node on average, in comparison to wildtype null plants (data not shown). Plants hemizygous for the Gm.FT2atransgene contained the highest number of pods per node over the lengthof the main stem. However, these effects were dependent on theparticular growing conditions including day length, etc. In general,experiments performed with soybean under longer day conditions tended toproduce greater differences between transgenic and null plants.

The dosage-dependent effects of transgenic Gm.FT2a expression were alsoobserved in field trial experiments. In a 2014 field trial, soybeanplants hemizygous for two Gm.FT2a events (Events 1 and 2 above) showedan average of about 2.68 pods per node on the main stem, and plantshomozygous for these events had about 1.40 pods per node on average,whereas null segregating plants had about 1.63 pods per node. In a 2013field trial, however, plants hemizygous for transgenic Gm.FT2a (Event 2)were found to have an average number of about 3.21 pods per node, ascompared to an average of about 3.05 pods per node in homozygous plantsand about 2.19 pods per node in null segregating plants. In another 2013micro plot experiment conducted at a different field location, plantshemizygous for the Gm.FT2a transgene (Event 1) were found to have about2.17 pods per node on average, as compared to an average of about 2.05pods per node in plants homozygous for the Gm.FT2a transgene (Event 2)and about 1.30 pods per node in null segregating plants. Thus, thenumber of pods per node on plants containing the Gm.FT2a transgene maydepend on a variety of factors including dosage of the FT transgene,environmental and field conditions, and perhaps differences in agronomicpractices. However, much like transgenic Gm.FT2a plants grown in thegreenhouse, homozygous and hemizygous Gm.FT2a transgenic plants grownunder field conditions often had fewer nodes on the main stem, shorteroverall plant height, and/or reduced branching in transgenic plants.Indeed, wild type plants typically had more branching and a greaternumber of total nodes per plant than hemizygous and homozygous Gm.FT2aplants.

Additional physiological data was collected from homozygous Gm.FT2atransgenic plants and wild type (WT) control plants grown in thegreenhouse under 14-hour long day conditions (see Table 2). These dataprovide an average of measurements taken from six Gm.FT2a transgenicplants for each event, or from eight wild type plants. The followingmatrices were collected for phenotypic characterization of these plants:Days to flowering at R1 (DOFR1); Days to R7 (DOR7); reproductiveduration in days from R1 to R7 (PDR1R7); number of branches per plant(BRPP); total fertile nodes on branches (FNBR); total fertile nodes perplant (FNLP); total fertile nodes on main stem (FNST); number of nodeson branches (NDBR); number of nodes on main stem (NDMS); number ofnodes/plant (NDPL); percent fertile nodes on branches (PFNB); percenttotal fertile nodes (PFNN); percent fertile nodes on main stem (PFNS);number of pods per plant (PDPP); number of pods on main stem (PODMS);number of pods on branches (PODBR); number of pods/node; seeds per plantat R8 (SDPPR8); and weight of 1000 seeds (SW1000). Each of thesemeasurements was taken at harvest unless another time point isspecified.

TABLE 2 Construct level phenotypic data for transgenic homozygousGm.FT2a and WT plants. WT pErecta::Gm.FT2a DOFR1 33.5 21.3 DOR7 106.992.9 PDR1R7 76.5 71.6 BRPP 20.1 1 FNBR 190.6 2 FNLP 214.6 15 FNST 24.014.3 NDBR 211.4 3 NDMS 33.4 15.3 NDPL 244.9 16.3 PDPP 575.8 61.2 PFNB90.4 75 PFNN 87.8 92.0 PFNS 71.4 92.9 PODBR 487.3 3 PODMS 88.4 60.2Pods/Node 2.4 3.8 SDPPR8 1319.6 122.1 SW1000 146 122.5 (grams)

Consistent with the observations noted above, homozygous Gm.FT2atransgenic plants experienced earlier floral induction than WT plants(DOFR1 about 21 days after planting, instead of about 33-34 days in wildtype plants). These measurements further showed that the number ofbranches (and other measurements related to branching, such as thenumber of nodes or pods on branches) was greatly reduced. Due to thetransgenic plants having a shorter stature with very little branching,the total numbers of nodes or pods per plant were also greatly reduced.However, the number of pods per node on the main stem was increased intransgenic plants (e.g., about 3.8 average pods/node) relative to wildtype null plants (e.g., about 2.4 pods/node).

Without being bound by any theory, the larger number of pods per nodeobserved with transgenic soybean plants expressing FT2a in the meristemduring vegetative stages of development may be caused at least in partby synchronization of early flowering with early secondary and/ortertiary raceme release and/or better resource utilization to producemore pod-producing flowers per node. Early FT expression in the meristem(see, e.g., FIGS. 3 and 4) may cause early release of the dormantinflorescence meristems to produce a greater number of racemes per nodeof the plant, such that a greater number of racemes produce matureflowers and fully developed pods at each node. However, subsequent FTexpression in reproductive tissues (see, e.g., FIGS. 5 and 6) mayterminate floral development of later developing flowers at each nodeleading to more efficient resource allocation to the earlier developingracemes, flowers and pods. In wild-type soybean plants, a much lowerpercentage of secondary and tertiary racemes produce flowers and fullydeveloped pods relative to primary racemes, and later developing flowersof the primary raceme typically do not produce mature flowers and/orfull-sized pods prior to abscission. Thus, it is theorized that morepods per node may be generated in plants expressing FT proteins in thevegetative meristem by synchronizing early flower development with earlyrelease of the lateral racemes at one or more node(s) of the plant. Withat least the pAt.Erecta promoter driving FT expression, later developingflowers (that may not otherwise produce fully developed or full-sizedpods) may also become terminated by later reproductive-stage expressionof FT to direct resources to the earlier developing flowers.

Example 4 Expression of Flowering Locus T Gene, Gm.FT2a, Under Controlof Alternative Vegetative Stage Promoters in Soybean

Based on the phenotypes observed in the preceding Example 3, twopromoters were also proposed to drive Gm.FT2a transgene expression thatwere considered vegetative-stage, leaf-preferred promoters: pAt.BLS (SEQID NO: 36) and pAt.ALMT6 (SEQ ID NO: 37). As used herein, a“leaf-preferred” promoter refers to a promoter that preferentiallyinitiates transcription of its associated gene in leaf tissues relativeto other plant tissues. Since FT is believed to function as a mobileflorigen, early FT expression during vegetative stages in peripheraltissues, such as in the leaf with a leaf-preferred or leaf-specificpromoter, may lead to phenotypes similar to the meristem-preferredpAt.Erecta:Gm.FT2a expression. It was further theorized that FTexpression with a vegetative leaf promoter might also attenuate thefloral induction signal, and thus mitigate the early terminationphenotypes observed with homozygous FT expression in the meristem, andincrease plant height and branching.

In these experiments, transformation vectors for pAt.ALMT6::Gm.FT2a andpAt.BLS::Gm.FT2a were constructed and used to transform a soybean lineby Agrobacterium-mediated transformation. Expression with the pAt.BLSpromoter has been shown to start in leaf primordia number 5 (p5) and isexpressed in the source leaf veins only until transition to flowering,and the pAt.ALMT6 promoter is also a vegetative leaf promoter withexpression at later developmental stages relative to pAt.BLS. See, e.g.,Efroni et al., “A Protracted and Dynamic Maturation Schedule UnderliesArabidopsis Leaf Development,” The Plant Cell 20(9): 2293-2306 (2008);and Shani et al., “Stage-Specific Regulation of Solanum lycopersicumLeaf Maturation by Class 1 KNOTTED1-LIKE HOMEOBOX Proteins,” The PlantCell 21(10): 3078-3092 (2009). Transgenic soybean plants were producedfor each of these vector constructs and characterized for phenotypes ingrowth chambers under 14-hour photoperiod conditions in comparison towild type plants. For each of the pAt.BLS construct, six transgenicevents were tested (5 plants per event), and for the pAt.ALMT6 promoter,seven transgenic events were tested (5 plants per event). For each ofthese constructs, control data was collected from five wild type plants.

The following matrices were collected for phenotypic characterization ofthese transgenic plants (Tables 3 and 4). The individual measurementsare as defined above, and phenotypic characterization was conducted onplants homozygous for the transgene.

TABLE 3 Construct level phenotypic data for pALMT6::Gm.FT2a and WTplants. WT pALMT6::FT2a DOFR1 35.2 38.8 DOR7 84.7 88.8 PDR1R7 49.5 50.0BRPP 7.7 8.9 FNBR 57.8 73.3 FNLP 69.7 85.0 FNST 12.0 11.7 NDBR 78.9 96.0NDMS 21.3 22.5 NDPL 100.2 118.5 PDPP 120.2 141.1 PFNB 73.2 76.8 PFNN71.7 72.1 PFNS 57.9 51.7 PODBR 91.8 118.1 PODMS 28.3 22.9 Pods/Node 1.41.2

TABLE 4 Construct level phenotypic data for pBLS::Gm.FT2a and WT plants.WT pBLS::FT2a DOFR1 31.3 35.2 DOR7 78.1 82.6 PDR1R7 46.9 47.5 BRPP 7.58.8 FNBR 65.7 81.2 FNLP 80.5 94.0 FNST 14.9 12.7 NDBR 72.2 95.6 NDMS21.9 22.3 NDPL 94.0 117.9 PDPP 137.0 148.1 PFNB 92.3 85.3 PFNN 87.4 80.1PFNS 68.1 57.3 PODBR 100.9 123.4 PODMS 36.1 24.8 Pods/Node 1.7 1.3

Transgenic plants expressing Gm.FT2a under the control of thealternative pAt.ALMT6 and pAt.BLS promoters were phenotypically moresimilar to wild type (WT) plants than pAT.Erecta::Gm.FT2a transgenicplants. Plants transformed with the pAt.ALMT6::Gm.FT2a and pAt.BLS::Gm.FT2a constructs had flowering times and vegetative growth traitssimilar to wild type control plants, perhaps with a slightly increasednumber of nodes on branches as compared to wild type plants (Tables 3and 4). These data may be interpreted to indicate that both the timingand location of transgenic FT expression are important for producingreproductive and yield-related traits or phenotypes that differ fromwild-type plants. Merely expressing a FT transgene during earliervegetative stages of development (e.g., in leaf tissues) may not besufficient to alter the reproductive or yield-related phenotypes of aplant (e.g., pods per node). Thus, according to embodiments of thepresent invention, a promoter operably linked to a florigenic FTtransgene may preferably be a meristem-specific or meristem-preferredpromoter in addition to driving expression during the vegetative stagesof plant development. However, when the expression profiles for theabove two leaf-preferred promoters were tested in soybean plants, no GUSstaining was observed in the developing leaf with the pAt.BLS promoter,and the pAt.ALMT6 promoter did not produce detectable GUS expression inthe leaf until late vegetative stages with much higher expression duringearly reproductive stages. Thus, it remains possible that expression ofFT transgenes in peripheral (leaf) tissues during early vegetativestages using different tissue-specific promoters may be sufficient insome cases to induce early flowering and/or cause other reproductive oryield-related traits or phenotypes, which may also depend on theparticular plant species tested.

Example 5 Identification of Protein Domains of FT Homologs by PfamAnalysis

Gm.FT2a orthologs were identified by sequence analysis and literaturereview, and a few examples of these FT homologs are listed in Table 5along with Gm.FT2a. These included other soybean FT genes as well as afew FT genes from other plant species. The amino acid sequences of theseFT proteins were analyzed to identify any Pfam protein domains using theHMMER software and Pfam databases (version 27.0). These FT proteinsequences (SEQ ID NOs: 2, 4, 6, 8, 10 and 12) were found to have thesame Pfam domain identified as a phosphatidyl ethanolamine bindingdomain protein (PEBP) having a Pfam domain name of “PBP N”, and a Pfamaccession number of PF01161. The location of the PBP N domains in eachof these FT protein sequences are also listed in Table 5. The locationof the PBP N domain in other FT proteins can be determined by sequencealignment. It is thus contemplated that any DNA sequence encoding atleast an FT protein comprising the PBP N domain may be used in arecombinant DNA molecule of the present invention, as long as thecorresponding FT protein has florigenic activity when ectopicallyexpressed in the meristem of a plant.

TABLE 5 Location of PBP_N (Pfam) domain in FT protein sequences. PROTEINDomain SEQ ID NO. Gene Name location 2 Gm.FT2a 28-162 4 Gm.FT5a 26-157 6Gm.FT2b 28-162 8 Zm.ZCN8 26-154 10 Nt.FT-like 25-159 12 Le.SFT 29-161

Example 6 Expression of FT Homologs Under Control of pAt.Erecta Promoterin Soybean

Additional transformation vectors containing other FT homologs (Table 6)under control of the pAt.Erecta promoter were constructed and used totransform soybeans via Agrobacterium-mediated transformation. Transgenicplants generated from these events were characterized for theirphenotypes in the greenhouse with a 14 to 14.5 hour natural daylightphotoperiod. For each construct, six events were tested (6 plants perevent). Six plants were also tested and averaged for wild type (WT)control plants. Different groups of experiments (A-E) were conducted asshown in Table 6 with separate wild type controls.

TABLE 6 List of constructs for some Gm.FT2a and its homologs with theirprotein sequences. Construct PROTEIN Description Gene Name SEQ ID NO.Testing Group pErecta:Gm.FT2a Gm.FT2a 2 A pErecta:Gm.FT2b Gm.FT2b 6 CpErecta:Gm.FT5a Gm.FT5a 4 E pErecta:Zm.ZCN8 Zm.ZCN8 8 BpErecta:Nt.FT-like Nt.FT-like 10 B pErecta:Le.SFT Le.SFT 12 D

The following matrices were collected for phenotypic characterization ofplants transformed with each of the constructs listed in Table 6 forexpressing other FT homologs with the pAt.Erecta promoter, in additionto data collected for the Gm.FT2a construct as described above. Theindividual measurements are as defined above, and phenotypiccharacterization of transformants was conducted on plants homozygous forthe transgene.

Phenotypic data was collected for plants expressing the Zm.ZCN8 andNt.FT-like transgenes under the control of the pAt.Erecta promoter (seeTables 7 and 8). Trait values for each Event in Tables 7 and 8 are anaverage of all plants tested containing the Event. A column is alsoprovided with an average of the Event values for each trait.

TABLE 7 Construct and event level phenotypic data for Zm.ZCN8 and WTplants. Aver- Event Event Event Event Event Event WT age 1 2 3 4 5 6DOFR1 33.5 28.6 29 29.2 27.5 27 30.7 28 DOR7 106.9 93.5 97.3 89.2 88.293.5 100.3 92.8 PDR1R7 76.5 64.1 69.8 60 59 59.5 71.7 64.8 BRPP 20.1 3.22.8 1.3 1.5 1.3 9.5 3 FNBR 190.6 26.9 32 8 7 2.3 95.3 17 FNLP 214.6 54.967.5 28 40.5 20.3 132.5 40.5 FNST 24.0 28.3 35.5 22 33.5 18 37.3 23.5NDBR 211.4 30.2 32.5 9 7.5 3.5 110.8 17.8 NDMS 33.4 30.5 36.3 24 34.3 2044.8 24 NDPL 244.9 60.3 68.8 30.8 41.8 23.5 155.5 41.8 PDPP 575.8 317.5498.3 144.8 319 76.3 658 208.8 PFNB 90.4 87.2 98.6 90.3 85.4 64.6 91.193.2 PFNN 87.8 93.1 98.1 92.3 97.0 86.5 88.5 96.4 PFNS 71.4 93.2 97.992.5 97.9 90.6 82.1 98.1 PODBR 487.3 105.4 162 19 18.5 3.3 384.5 45.3PODMS 88.4 212.9 336.3 130.5 300.5 73 273.5 163.5 Pods/ 2.4 5.5 7.2 4.67.7 3.2 4.9 5.2 Node SDPP8 1319.6 564.7 961 200.5 562 136.8 1166.3 361.5SW1000 146 108.9 102.9 127.4 105.3 82.9 116.6 117.9 (grams)

TABLE 8 Construct and event level phenotypic data for Nt.FT-like and WTplants. Aver- Event Event Event Event Event Event WT age 1 2 3 4 5 6DOFR1 33.5 31.5 39.3 27.7 25.3 29 37.2 30.7 DOR7 106.9 93.9 115.8 90.780.7 83.7 102.2 90.2 PDR1R7 76.5 62.3 76.4 63 55.3 54.7 65 59.5 BRPP20.1 9.8 20 8.3 2.3 5.3 17 6 FNBR 190.6 108.7 190.5 95.3 11 54.3 22378.3 FNLP 214.6 131.4 212.3 118 29.5 77.8 248 103 FNST 24.0 23.2 21.822.8 21.3 23.5 25 24.8 NDBR 211.4 128.7 281.8 97 11 54.5 247.7 80.5 NDMS33.4 28.9 33.8 27 23.3 24.8 35.7 28.8 NDPL 244.9 157.1 315.5 124 31.579.3 283.3 109.3 PDPP 575.8 462.1 638 511.3 150.8 296 745 431.5 PFNB90.4 92.5 68.0 98.6 100 99.7 91.3 97.2 PFNN 87.8 89.6 67.6 95.4 93.398.3 88.4 94.2 PFNS 71.4 81.9 64.7 83.3 91.3 95.2 70.6 86.2 PODBR 487.3326.3 529 342.3 22.7 147 633.3 283.5 PODMS 88.4 136.7 109 169 133.8 149111.7 148 Pods/ 2.4 3.6 2.0 4.3 4.9 3.8 2.7 4.0 Node SDPPR8 1319.6 928.71359.8 965.5 382.7 591.5 1714.3 558.7 SW1000 146 149.0 143.7 121.2 133.8179.3 142.2 174.0 (grams)

Transgenic soybean plants expressing the Zm.ZCN8 and Nt.FT-like proteinsflowered earlier than wild type control plants and had an increasednumber of pods per node (similar to plants expressing the Gm.FT2atransgene). Indeed, soybean plants expressing the Zm.ZCN8 and Nt.FT-liketransgenes had several phenotypes similar to the Gm.FT2a transgenicplants, including reduced number of days to flowering (DOFR1), reducednumber of branches (BRPP), fewer nodes per plant (NDPL), fewer nodes onbranches (NDBR), reduced number of pods per plant (PDPP), and fewer podson branches (PODBR), along with an increase in the number of pods pernode and a decrease in the number of seeds per plant (Tables 7 and 8),relative to wild type controls. However, several of the negativephenotypes observed in homozygous Gm.FT2a plants were less pronounced inthe Zm.ZCN8 and Nt.FT-like expressing transgenic plants. Overall, plantsexpressing the Zm.ZCN8 transgene had shorter plant height and lessbranching but more pods per node on the main stem (FIGS. 12A and 12B).Similarly, plants expressing the Nt.FT-like transgene had shorter plantheight, reduced branching and increased pods per node on the main stem(FIGS. 13A and 13B), relative to wild type control plants.

Two transgenic Zm.ZCN8 events and four Nt.FT-like events from above werealso tested in 2015 field trials at two different locations. Phenotypicdata was collected for plants expressing Zm.ZCN8 and Nt.FT-liketransgenes under the control of the pAt.Erecta promoter (Tables 9 and10). Events 1 and 2 in Table 9 correspond to Events 2 and 3 in Table 7,and Events 1-4 in Table 10 correspond to Events 1-4 in Table 8,respectively. Except for days to flowering at R1 (DOFR1) andreproductive duration in days from R1 to R8 (PDR1R8), all phenotypicmeasurements were derived based on data collected from two locations.Similar to the observations in the greenhouse, transgenic soybean plantsexpressing Zm.ZCN8 and Nt.FT-like proteins also flowered earlier thanwild-type control plants in the field. The Zm.ZCN8 transgenic plants hadan increased number of pods per node, while the Nt.FT-like plants didnot clearly show increased pods per node in the field trial.

TABLE 9 Phenotypic data from 2015 field trial for Zm.ZCN8 and WT plants.WT Average Event 1 Event 2 DOFR1* 42.4 27.9 28.0 27.7 DOR8 110.7 95.092.0 98.0 PDR1R8* 65.7 67.1 63.5 70.7 BRPP 2.6 0.1 0.2 0.0 NDBR 9.7 0.30.5 0.1 NDMS 18.3 13.6 12.5 14.7 NDPL 28.0 13.9 13.0 14.8 PDPP 44.2 35.130.1 40.0 TPBR 9.5 0.3 0.5 0.1 PODMS 34.7 34.7 29.5 39.9 Pods/Node 1.62.5 2.3 2.6 SDPPR8 99.9 67.6 54.7 80.5 SW1000 5.1 4.1 3.8 4.3 (ounces)(*single location data)

TABLE 10 Phenotypic data from 2015 field trial for Nt.FT-like and WTplants. Aver- Event Event Event Event WT age 1 2 3 4 DOFR1* 42.4 38.042.5 26.8 26.8 25.8 DOR8 110.7 93.3 111.3 88.2 86.6 87.1 PDR1R8* 65.763.1 66.8 62.2 60.3 63.0 BRPP 2.6 0.7 2.4 0.1 0.2 0.1 NDBR 9.7 2.7 9.20.5 0.8 0.3 NDMS 18.3 11.5 18.3 9.9 7.6 10.1 NDPL 28.0 14.2 27.5 10.48.5 10.4 PDPP 44.2 23.5 43.0 18.9 11.6 20.3 TPBR 9.5 2.6 8.4 0.5 0.8 0.5PODMS 34.7 20.9 34.6 18.5 10.8 19.8 Pods/ 1.6 1.6 1.6 1.8 1.4 1.7 NodeSDPPR8 99.9 49.9 98.6 36.3 25.0 39.7 SW1000 5.1 4.5 5.1 4.4 4.6 4.0(ounces) (*single location data)

Additional phenotypic data was collected for plants expressing theGm.FT2b transgene under the control of the pAt.Erecta promoter (Table11).

TABLE 11 Construct and event level phenotypic data for Gm.FT2b and WTplants. Aver- Event Event Event Event Event Event WT age 1 2 3 4 5 6DOFR1 43.7 34.6 41.2 34.3 22.7 33.2 37.2 39.3 DOR7 105.9 100.4 100.5100.3 99.8 100.3 98.7 102.8 PDR1R7 62.2 65.8 59.3 66 77.2 67.2 61.5 63.5BRPP 13.4 4.7 7 5 1.7 3.3 3.7 7.7 FNBR 103.8 32.4 52 29.7 12 30.7 21.748.7 FNLP 125.0 46.6 68.7 41.3 24.3 45 37.3 63 FNST 21.2 14.2 16.7 11.712.3 14.3 15.7 14.3 NDBR 108.4 34.2 54 30.3 12.7 33.7 24.7 50 NDMS 30.218.0 18.3 15.3 15 19 19.3 21 NDPL 138.7 52.2 72.3 45.7 27.7 52.7 44 71PDPP 387.4 143.0 167 140 96 145.7 108.7 200.7 PFNB 95.5 94.6 96.4 97.796.8 91.0 87.7 97.7 PFNN 90.1 89.1 94.9 90.3 88.0 86.0 86.1 89.4 PFNS69.7 79.2 91.5 74.5 82.5 77.0 81.1 68.7 PODBR 284.9 90.2 109.3 96 4394.7 55.3 143 PODMS 102.5 52.7 57.7 44 53 51 53.3 57.7 Pods/ 2.8 2.7 2.33.1 3.5 2.8 2.5 2.8 Node SDPPR8 1159.3 322.3 411.3 292.3 195.3 346.7 245443.3 SW1000 174.0 154.0 170.4 156.7 154.1 155 130.2 157.8 (grams)

Transgenic soybean plants expressing the Gm.FT2b transgene floweredearlier and had less branching than wild type control plants. Gm.FT2bexpressing soybean plants had a reduced number of days to flowering(DOFR1), reduced number of branches (BRPP), fewer nodes per plant(NDPL), fewer nodes on branches (NDBR), reduced number of pods per plant(PDPP), and fewer pods on branches (PODBR) (Table 9). However,transgenic Gm.FT2b plants did not show an increase in the number of podsper node. Overall, plants expressing the Gm.FT2b transgene had shorterplant height and less branching relative to wild type control plants(FIG. 14). Transgenic soybean plants expressing four different events ofthe Gm.FT2b transgene were also tested in 2015 field trials. Phenotypicdata was collected for plants expressing the Gm.FT2b transgene under thecontrol of the pAt.Erecta promoter (Table 12). Events 1-4 in Table 11correspond to Events 3, 2, 1, and 4 in Table 12, respectively. Similarto the observations in the greenhouse, Gm.FT2b expressing soybean plantsshowed a reduced number of days to flowering (DOFR1) in the field. Theother phenotypic measurements also exhibited similar traits as observedin the greenhouse relative to wild-type control plants.

TABLE 12 Phenotypic data from 2015 field trial for Gm.FT2b and WTplants. Aver- Event Event Event Event WT age 1 2 3 4 DOFR1 41.9 37.338.3 38.3 36.3 36.2 DOR8 115.4 109.2 111.1 110.6 110.6 104.6 PDR1R8 73.571.8 75.0 72.1 74.1 66.1 SDPPR8 188.5 95.5 99.4 81.1 117.7 83.6 SW1000153.4 137.3 144.0 129.6 134.4 141.5 (grams)

Additional phenotypic data was collected from plants expressing theLe.SFT transgene under the control of the pAt.Erecta promoter (Table13).

TABLE 13 Construct and event level phenotypic data for Le.SFT and WTplants. Aver- Event Event Event Event Event Event WT age 1 2 3 4 5 6DOFR1 42.9 41.4 30 44.4 30.7 28 60.2 55 DOR7 108.6 103.8 90.7 106.2 9991.5 116.5 119 PDR1R7 65.5 64.0 60.2 71 67 65 56.4 64.2 BRPP n/a n/a n/an/a n/a n/a n/a n/a FNBR 131.5 37.1 2.7 125.2 4.7 1 47.7 41.3 FNLP 156.850.7 15.7 142.6 18.1 18.3 56.7 52.7 FNST 25.3 13.7 13 17.7 13.7 17.3 911.3 NDBR 140.2 38.9 3 129.4 5.4 1 51 43.7 NDMS 32.4 17.9 16 25.8 16.321 12.3 16.3 NDPL 172.4 56.7 19 154.7 21.2 22 63.3 60 PDPP 473.3 201.353.7 432.3 69.8 85.7 279.3 287 PFNB 94.1 94.0 100 96.9 83.3 100 92.990.7 PFNN 90.4 86.4 82.5 92.3 83.7 83.5 88.8 87.6 PFNS 77.1 76.5 81.269.5 84.0 82.8 72.7 68.6 PODBR 366.2 141.8 3.7 361.5 15.5 1.3 238.7230.7 PODMS 114 60.3 50 73.6 57.1 84.3 40.7 56.3 Pods/ 2.7 3.6 2.8 2.83.3 3.9 4.4 4.8 Node SDPPR8 1247.4 476.0 136.7 1036 148.5 183 655 697SW1000 167.7 153.2 170.0 182.8 157.5 148.5 131.5 128.8 (grams)

Overall, soybean plants expressing the Le.SFT transgene had shorterplant height with less branching (FIG. 15) and an increased number ofpods per node on average relative to wild type plants (Table 13).However, these effects were variable and event-specific. For example,Events 1, 3 and 4 displayed early flowering (DOFR1), while other eventswere neutral or actually had delayed flowering. In addition, some of theLe.SFT transgenic events showed increased pods per node on average tovarying extents, while a couple of the events were neutral in terms ofthe average number of pods per node. Interestingly, two of the events(Events 5 and 6) had the greatest number of pods per node on averagedespite having a delay in flowering.

Additional phenotypic data was collected from plants expressing theGm.FT5a transgene under the control of the pAt.Erecta promoter (Table14).

TABLE 14 Construct and event level phenotypic data for Gm.FT5a and WTplants. Aver- Event Event Event Event Event WT age 1 2 3 4 5 DOFR1 48.229.9 32.2 29 28.6 29.2 30.5 DOR7 110 92.5 96.6 90.4 91 92.8 91.8 PDR1R761.8 62.7 64.4 61.4 62.4 63.6 61.3 BRPP 12.4 2.5 7 1.7 1 1.3 1.7 FNBR105.6 7.3 20.3 4.7 3 4 4.3 FNLP 126.5 24.5 41.7 20 18.7 19.3 22.7 FNST20.9 17.2 21.3 15.3 15.7 15.3 18.3 NDBR 108.6 7.5 21 5 3 4 4.3 NDMS 2917.7 22 15.7 16.3 16 18.7 NDPL 137.6 25.2 43 20.7 19.3 20 23 PDPP 304.3131.9 214.7 111 100.3 104.3 129.3 PFNB 97.2 98.0 97.3 93.3 100 100 100PFNN 98.1 97.0 95.9 90 99.1 100 100 PFNS 72.1 97.0 97.1 98.1 96.1 95.897.9 PODBR 233.4 16.5 60.5 8 4 6 4 PODMS 75.1 108.6 159 98.5 95 92.5 98Pods/ 2.2 5.2 5.0 5.4 5.2 5.2 5.6 Node SDPPR8 778.8 271.7 516 232.7175.3 182.3 252 SW1000 151.6 126.0 143.7 122.4 122.2 121.8 116.8 (grams)

Transgenic soybean plants expressing the Gm.FT5a transgene floweredsignificantly earlier than wild type control plants and had an increasednumber of pods per node (similar to plants expressing the Gm.FT2atransgene). Indeed, soybean plants expressing the Gm.FT5a transgene hadseveral phenotypes (similar to the Gm.FT2a transgenic plants), includingreduced number of days to flowering (DOFR1), reduced number of branches(BRPP), fewer nodes per plant (NDPL), fewer nodes on branches (NDBR),reduced number of pods per plant (PDPP), and fewer pods on branches(PODBR), along with an increase in the number of pods per node and adecrease in the number of seeds per plant (Table 14). Overall, plantsexpressing the Gm.FT2a transgene had shorter plant height and lessbranching, but more pods per node (particularly on the main stem)relative to wild type control plants (FIG. 16).

Without being bound by any theory, these data support a model of FToverexpression acting in a dosage-dependent manner with the degree orextent of associated phenotypes (e.g., early flowering, increase in podsper node, and altered plant architecture) depending on (i) the level andtiming of FT expression, (ii) tissue specificity of FT expression, and(iii) the relative activity and target specificity of the particular FTprotein being expressed. For example, expression of the FT proteinorthologs from other plant species in soybean may produce a moreattenuated effect relative to overexpression of an endogenous FT protein(Gm.FT2a) in soybean, which may result from the non-native FT proteinhomologs having a lower activity in soybean. However, expression of somenative FT proteins may not produce significant phenotypic effects ifthey have a different or specialized role in their native state orcontext. Different FT proteins may also act on different tissue targetsand receptors and thus have differential effects on the various plantarchitecture and flowering traits and phenotypes.

Regardless of the activity level of the particular FT homolog, alteredreproductive and plant architecture phenotypes appear to correlate withthe timing and location of FT expression. Vegetative-stage expression ofFT transgenes may be necessary to induce early flowering and/or causeincreased numbers of floral meristems, flowers, pods, etc., per node ofthe plant. Indeed, FT expression in meristematic tissues duringvegetative stages of development is shown with proper dosing of the FTtransgene to cause reproductive changes in plants leading to increasednumbers of flowers, pods, and/or seeds per node. In contrast, expressionof a Gm.FT2a transgene under the control of leaf-preferred promotersproduced very little, if any, phenotypic changes, relative to wild typeplants. These data indicate that both the timing, and tissue specificity(or tissue preference), of FT expression are important factors thataffect reproductive and/or yield-related phenotypic changes intransgenic plants.

The present data suggest that different FT proteins may have differentactivity levels and/or target specificities despite being expressedusing the same pErecta promoter. While several constructs expressingGm.FT2a, Zm.ZCN8, Nt.FT-like, and Gm.FT5a each caused early floweringand termination in addition to an increased number of pods per node,other constructs expressing Gm.FT2b and Le.SFT had different correlativeeffects on flowering. Expression of Gm.FT2b did cause early floweringand termination of plants but without a significant increase in thenumber of pods per node. On the other hand, Le.SFT expression showedincreased pods per node and early termination despite a delay inflowering. Interestingly, increased numbers of pods per node intransgenic FT plants did not correlate with an extended reproductiveduration (PDR1R7) and was not always aligned with early flowering(DOFR1) as noted above. These data suggest that reproductive changes inresponse to vegetative-stage expression of FT proteins in the meristemmay operate through one or more independent mechanisms or pathways.Increased numbers of pods per node in transgenic FT plants may depend onthe number of inflorescent and floral meristems induced from vegetativemeristems at each node, which may occur independently of flowering timeand/or reproductive duration. As noted above, however, reproductiveduration may not necessarily correlate with the duration of flowering.

Example 7 Identification of Additional Vegetative-Stage MeristemPromoters

Having observed phenotypic effects with expression of Gm.FT2a under thecontrol of a vegetative-stage, meristem-preferred promoter, pAt.Erecta,it is contemplated that other vegetative-stage, meristem-preferred (ormeristem-specific) promoters may be used to drive expression of FTproteins to cause reproductive or yield-related traits or phenotypes inplants, such as increased number of pods per node (and/or per plant ormain stem). Using the characterized expression pattern of the pAt.Erectapromoter (see Example 2), other vegetative-stage, meristem-preferred (ormeristem-specific) promoters were identified from soybean, potato andArabidopsis. Two bioinformatic approaches were utilized to identifycandidate genes from other dicotyledonous species including, forexample, Arabidopsis, soybean, Medicago, potato and tomato, havingsimilar expression profiles to pAt.Erecta: BAR Espressolog andExpression Angler. See, e.g., BAR expressolog identification: expressionprofile similarity ranking of homologous genes in plant species, PlantJ71(6): 1038-50 (2012); and Toufighi, K et al., “The Botany ArrayResource: e-Northerns, Expression Angling, and promoter analyses,” PlantJ 43(1): 153-163 (2005). The promoter sequences from these genes arethus proposed for use in expressing FT transgenes according toembodiments of the present invention.

Examples of gene promoters identified by this analysis include thefollowing: four receptor like kinase (RLK) genes from soybean, includingGlyma10g38730 (SEQ ID NO: 23), Glyma09g27950 (SEQ ID NO: 24),Glyma06g05900 (SEQ ID NO: 25), and Glyma17g34380 (SEQ ID NO: 26).Additional examples include receptor like kinase (RLK) gene promotersfrom potato, PGSC0003DMP400032802 (SEQ ID NO: 27) andPGSC0003DMP400054040 (SEQ ID NO: 28). It is possible that these RLKgenes may be related structurally and/or functionally to Erecta andErecta-like genes from Arabidopsis and other species since they are alsoRLK genes. Other vegetative stage, meristem-preferred promoters fromArabidopsis genes include the following: At.MYB17 (At.LMI2; At3g61250)(SEQ ID NO: 31), Kinesin-like gene (At5g55520) (SEQ ID NO: 32),AP2/B3-like genes including ALREM17 (SEQ ID NO: 33) or ALREA119, andErecta-like 1 and 2 genes, At.Erl1 (SEQ ID NO: 34) and At.Erl2 (SEQ IDNO: 35). Each of these promoters and similar functional sequences may beoperatively linked to a FT gene to cause ectopic expression of FT genesin one or more meristem(s) of plants at least during vegetative stage(s)of development.

With regard to the At.MYB17 (At.LMI2) gene, see Pastore, J L et al.,“LATE MERISTEM IDENTITY 2 acts together with LEAFY to activateAPETALA1,” Development 138: 3189-3198 (2011), the entire contents anddisclosure of which are incorporated herein by reference. With regard tothe Kinesin-like gene, see Fleury, D et al., “The Arabidopsis thalianaHomolog of Yeast BREI Has a Function in Cell Cycle Regulation duringEarly Leaf and Root Growth,” Plant Cell, 19(2): 417-432 (2007), theentire contents and disclosure of which are incorporated herein byreference. With regard to the REM17 and REM19 Arabidopsis genes, seeMantegazza, 0 et al., “Analysis of the Arabidopsis REM gene familypredicts functions during flower development,” Ann Bot 114(7): 1507-1515(2014), the entire contents and disclosure of which are incorporatedherein by reference. Further, with regard to the At.Erl2 gene, see“Special Issue: Receptor-like Kinases,” JIPB 55(12): 1181-1286 (2013),and particularly Shpak, E., “Diverse Roles of ERECTA Family Genes inPlant Development,” JIPB 55(12): 1251-1263 (2013), the entire contentsand disclosures of which are incorporated herein by reference.

Example 8 Expression of Flowering Locus T Gene, Gm.FT2a, Under Controlof a Paterll Promoter Alters Flowering Time and Pods Per Node in Soybean

A transformation vector containing Gm.FT2a under control of thevegetative stage, meristem-preferred pAt.Erl1 promoter (SEQ ID NO: 34)was constructed and used to transform soybeans viaAgrobacterium-mediated transformation. Transgenic plants generated fromthese events were characterized for their phenotypes in the greenhousewith a 14 to 14.5 hour natural daylight photoperiod. For eachpAt.Er11:Gm.FT2a construct, six events were tested (6 plants per event).Six plants were also tested and averaged for wild type (WT) controlplants. The following matrices were collected for phenotypiccharacterization of these plants and expressed as an average for eachEvent as well as the wild type plants (see Table 15). A column providingan average for all the Events per trait is further provided.

TABLE 15 Phenotypic data for pAt.Erl1:Gm.FT2a and WT plants. Aver- EventEvent Event Event Event Event WT age 1 2 3 4 5 6 DOFR1 46.1 32.6 40.032.3 34.0 29.3 28.2 31.8 DOR7 115.1 99.0 109.7 99.0 99.0 93.0 91.7 101.7PDR1R7 69.0 66.4 69.7 66.7 65.0 63.7 63.5 69.8 BRPP 23.5 7.4 16.0 6.09.7 1.3 4.3 7.3 NDBR 277.6 80.8 215.7 51.7 139.3 3.3 18.0 56.7 NDMS 29.832.3 30.7 33.7 32.7 30.7 32.7 33.3 NDPL 307.4 113.0 246.3 85.3 172.034.0 50.7 90.0 PDPP 605.8 346.4 447.3 349.7 493.7 240.7 194.7 352.3PODBR 503.1 173.7 332.3 164.7 323.3 8.3 42.7 171.0 PODMS 103.0 172.7115.0 185.0 170.3 232.3 152.0 181.3 Pods/ 1.9 4.0 2.1 4.1 2.9 7.1 3.84.0 Node SDPPR8 1290.0 747.5 1129.0 603.5 881.0 577.0 432.3 862.3 SW1000157.6 157.3 187.7 142.6 173.8 144.3 144.9 150.3 (grams)

Transgenic soybean plants expressing a pAt.Er11::Gm.FT2a constructflowered earlier than wild type control plants and had an increasednumber of pods per node (similar to plants expressing the Gm.FT2atransgene under control of the pAt.Erecta promoter). Indeed, soybeanplants expressing pAt.Er11:Gm.FT2a had several phenotypes similar to thepAt.Erecta:Gm.FT2a transgenic plants, including reduced number of daysto flowering (DOFR1), reduced number of days to R7 (DOR7), reducednumber of branches (BRPP), fewer nodes per plant (NDPL), a reducednumber of pods per plant (PDPP), along with an increase in the number ofpods per node (Table 15), relative to wild type control plants. However,several phenotypes observed in pAt.Erecta::Gm.FT2a plants, such asnumber of pods on main stem (PODMS), number of pods on branches (PODBR),and weight of 1000 seeds (SW1000), were less pronounced inthepAtEr11::Gm.FT2a expressing transgenic plants.

The expression pattern for the Arabidopsis Erecta-like 1 promoter(pAt.Erl1) in soybean as measured by GUS staining is more restrictedthan the expression pattern of pAt.Erecta in soybean as described above.pAt.Erl1 drives GUS expression in vegetative axillary meristems and inearly floral meristems derived from axillary tissue. However, GUSstaining is not observed in the shoot apical meristem at any stage whereit can be distinguished from other meristematic tissues of thedeveloping plant. Expression of the GUS reporter under the control ofthe pAt.Erl1 promoter is not observed in leaf tissue, stem or root atany stage (data not shown). Given that FT expression under the controlof either the pAt.Erecta or pAt.Erl1 promoter induced early floweringand increased pods per node, vegetative expression of an FT transgene ator near the meristem(s) of a plant may generally be sufficient to inducethese reproductive and yield-related phenotypes or traits.

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments are possiblewithout departing from the spirit and scope of the present disclosure asdescribed herein and in the appended claims. Furthermore, it should beappreciated that all examples in the present disclosure are provided asnon-limiting examples.

What is claimed is:
 1. A recombinant DNA construct comprising apolynucleotide sequence encoding a florigenic FT protein operably linkedto a vegetative stage promoter.
 2. The recombinant DNA construct ofclaim 1, wherein the florigenic FT protein comprises an amino acidsequence having at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or least 99% identity to a sequence selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, or afunctional fragment thereof.
 3. The recombinant DNA construct of claim2, wherein the florigenic FT protein further comprises one or more ofthe following amino acids: a tyrosine or other uncharged polar ornonpolar residue at the amino acid position of the florigenic FT proteincorresponding to amino acid position 85 of SEQ ID NO: 14; a leucine orother nonpolar residue at the amino acid position of the florigenic FTprotein corresponding to amino acid position 128 of SEQ ID NO: 14; and atryptophan or other large nonpolar residue at the amino acid position ofthe florigenic FT protein corresponding to amino acid position 138 ofSEQ ID NO:
 14. 4. The recombinant DNA construct of claim 2, wherein theflorigenic FT protein does not have one or more of the following aminoacids: a histidine at the amino acid position corresponding to a lysineor arginine at the amino acid position corresponding to position 85 ofSEQ ID NO: 14; a lysine or arginine at the amino acid positioncorresponding to position 128 of SEQ ID NO: 14; and a serine, asparticacid, glutamic acid, lysine or arginine at the amino acid positioncorresponding to position 138 of SEQ ID NO:
 14. 5. The recombinant DNAconstruct of claim 2, wherein the florigenic FT protein comprises anamino acid sequence selected from the group consisting of SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, and 20, or a functional fragment thereof.6. The recombinant DNA construct of claim 1, wherein the polynucleotidesequence is at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity to a sequence selected from thegroup consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19.7. The recombinant DNA construct of claim 1, wherein the vegetativestage promoter is a meristem-preferred or meristem-specific promoter. 8.The recombinant DNA construct of claim 1, wherein the promoter comprisesa polynucleotide sequence that is at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or least 99% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNOs: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35, or afunctional portion thereof.
 9. The recombinant DNA construct of claim 1,wherein the promoter comprises the pAt.Erecta promoter of SEQ ID NO: 21,or a functional portion thereof
 10. The recombinant DNA construct ofclaim 9, wherein the promoter comprises the truncated pAt.Erectapromoter of SEQ ID NO: 22 or SEQ ID NO:
 38. 11. The recombinant DNAconstruct of claim 1, wherein the promoter comprises the pAt.Erl 1promoter of SEQ ID NO: 34, or a functional portion thereof.
 12. A DNAmolecule or vector comprising the recombinant DNA construct of claim 1.13. A plasmid vector for Agrobacterium-mediated transformationcomprising the recombinant DNA construct of claim
 1. 14. A donortemplate molecule for site-directed integration comprising therecombinant DNA construct of claim
 1. 15. A transgenic plant comprisingan insertion of the recombinant DNA construct of claim 1 into the genomeof the transgenic plant.
 16. The transgenic plant of claim 13, whereinthe transgenic plant is homozygous for the insertion of the recombinantDNA construct.
 17. The transgenic plant of claim 15, wherein thetransgenic plant is hemizygous for the insertion of the recombinant DNAconstruct.
 18. The transgenic plant of claim 15, wherein the transgenicplant is a short day plant.
 19. The transgenic plant of claim 15,wherein the transgenic plant is a dicotyledonous plant.
 20. Thetransgenic plant of claim 19, wherein the transgenic plant is aleguminous plant.
 21. The transgenic plant of claim 20, wherein thetransgenic plant is soybean.
 22. The transgenic plant of claim 21,wherein the transgenic soybean plant produces more pods per node than acontrol plant not having the recombinant DNA construct.
 23. Thetransgenic plant of claim 15, wherein the transgenic plant produces moreflowers per node than a control plant not having the recombinant DNAconstruct.
 24. The transgenic plant or part thereof of claim 15, whereinthe transgenic plant produces more bolls, siliques, fruits, nuts or podsper node of the transgenic plant than a control plant not having therecombinant DNA construct.
 25. The transgenic plant or part thereof ofclaim 15, wherein the transgenic plant flowers earlier than a controlplant not having the recombinant DNA construct.
 26. The transgenic plantor part thereof of claim 15, wherein the transgenic plant has morefloral racemes per node than a control plant not having the recombinantDNA construct.
 27. A transgenic plant part comprising the recombinantDNA construct of claim
 1. 28. The transgenic plant part of claim 27,wherein the transgenic plant part is one of the following: a seed,fruit, leaf, cotyledon, hypocotyl, meristem, embryo, endosperm, root,shoot, stem, pod, flower, infloresence, stalk, pedicel, style, stigma,receptacle, petal, sepal, pollen, anther, filament, ovary, ovule,pericarp, phloem, or vascular tissue.
 29. A method for producing atransgenic plant, comprising (a) transforming at least one cell of anexplant with a recombinant DNA construct comprising a polynucleotidesequence encoding a florigenic FT protein operably linked to avegetative stage promoter; and (b) regenerating or developing thetransgenic plant from the transformed explant.
 30. The method of claim29, wherein the vegetative stage promoter of the recombinant DNAconstruct is a meristem-preferred or meristem-specific promoter.
 31. Themethod of claim 29, further comprising: (c) selecting a transgenic planthaving one or more of the following traits or phenotypes: earlierflowering, longer reproductive or flowering duration, increased numberof flowers per node, increased number of floral racemes per node,increased number of pods, bolls, siliques, fruits, or nuts per node, andincreased number of seeds per node, as compared to a control plant nothaving the recombinant DNA construct.
 32. The method of claim 29,wherein the transforming step (a) is carried out viaAgrobacterium-mediated transformation or microprojectile bombardment ofthe explant.
 33. The method of claim 29, wherein the transforming step(a) comprises site-directed integration of the recombinant DNAconstruct.
 34. A method of planting a transgenic crop plant, comprising:planting the transgenic crop plant at a higher density in the field,wherein the transgenic crop plant is transformed with a recombinant DNAconstruct comprising a polynucleotide sequence encoding a florigenic FTprotein operably linked to a vegetative stage promoter.
 35. The methodof claim 34, wherein the vegetative stage promoter is ameristem-preferred or meristem-specific promoter.
 36. The method ofclaim 34, wherein the transgenic crop plant is soybean, and whereinabout 150,000 to 250,000 seeds of the transgenic soybean plant areplanted per acre.
 37. The method of claim 34, wherein the transgeniccrop plant is cotton, and wherein about 48,000 to 60,000 seeds of thetransgenic cotton plant are planted per acre.
 38. The method of claim34, wherein the transgenic crop plant is canola, and wherein about450,000 to 680,000 seeds of the transgenic canola plant are planted peracre.